Compositions that specifically bind to colorectal cancer cells and methods of using the same

ABSTRACT

A unique transcription product, CRCA-1, and alternative translation products generated thereform, are disclosed. The transcript and its translation products are markers for colorectal cells. Screening and diagnostic reagents, kits and methods for metastasized colorectal cancer are disclosed ars are reagents, kits and methods for identifying adenocarcinomas as colorectal in origin. Compounds, composotions and methods of treating patients with metastasized colorectal cancer and for imaging metastasized colorectal tumors in vivo are disclosed. Compositions and methods for delivering active compounds such as gene therapeutics and antisense compounds to colorectal cells are disclosed. Vaccines compositions and methods of for treating and preventing metastasized colorectal cancer are disclosed.

FIELD OF THE INVENTION

[0001] The present invention relates to in vitro diagnostic methods fordetecting colorectal cancer cells, to kits and reagent for performingsuch methods. The present invention relates to compounds and methods forin vivo imaging and treatment of colorectal tumors. The presentinvention relates to methods and compositions for making and usingtargeted gene therapy, antisense and drug compositions. The presentinvention relates to prophylactic and therapeutic anti-colorectal cancervaccines and compositions and methods of making and using the same.

BACKGROUND OF THE INVENTION

[0002] Colorectal cancer is the third most common neoplasm worldwide.The mortality rate of newly diagnosed large bowel cancer approaches 50%and there has been little improvement over the past 40 years. Most ofthis mortality reflects local, regional and distant metastases.

[0003] Surgery is the mainstay of treatment for colorectal cancer butrecurrence is frequent. Colorectal cancer has proven resistant tochemotherapy, although limited success has been achieved using acombination of 5-fluorauracil and levamisole. Surgery has had thelargest impact on survival and, in some patients with limited disease,achieves a cure. However, surgery removes bulk tumor, leaving behindmicroscopic residual disease which ultimately results in recrudescence.

[0004] Early detection of primary, metastatic, and recurrent disease cansignificantly impact the prognosis of individuals suffering fromcolorectal cancer. Large bowel cancer diagnosed at an early stage has asignificantly better outcome than that diagnosed at more advancedstages. Similarly, diagnosis of metastatic or recurrent disease earlierpotentially carries with it a better prognosis.

[0005] Although current radiotherapeutic agents, chemotherapeutic agentsand biological toxins are potent cytotoxins, they do not discriminatebetween normal and malignant cells, producing adverse effects anddose-limiting toxicities. Over the past decade, a novel approach hasbeen employed to more specifically target agents to tumor cells,involving the conjugation of an active agent to molecules which bindspreferentially to antigens that exist predominantly on tumor cells.These conjugates can be administered systemically and specifically bindto the targeted tumor cells. Theoretically, targeting permits uptake bycells of cytotoxic agents at concentrations which do not produce serioustoxicities in normal tissues. Also, selective binding to targeted tumorcells facilitates detection of occult tumor and is therefore useful indesigning imaging agents. Molecular targeting predominantly has employedmonoclonal antibodies generated to antigens selectively expressed ontumor cells.

[0006] Immunoscintigraphy using monoclonal antibodies directed attumor-specific markers has been employed to diagnose colorectal cancer.Monoclonal antibodies against carcinoembryonic antigen (CEA) labelledwith ⁹⁹Technetium identified 94% of patients with recurrent tumors.Similarly, ¹¹¹Indium-labelled anti-CEA monoclonal antibodiessuccessfully diagnosed 85% of patients with recurrent colorectalcarcinoma who were not diagnosed by conventional techniques.¹²⁵Iodine-labelled antibodies have been effective in localizing morethan 80% of the pathologically-confirmed recurrences by intraoperativegamma probe scanning.

[0007] Monoclonal antibodies have also been employed to target specifictherapeutic agents in colorectal cancer. Preclinical studiesdemonstrated that anti-CEA antibodies labelled with ⁹⁰Yttrium inhibitedhuman colon carcinoma xenografts in nude mice. Antibodies generated tocolorectal cancer cells and coupled to mitomycin C or neocarzinostatindemonstrated an anti-tumor effect on human colon cancer xenografts innude mice and 3 patients with colon cancer. Similar results in animalswere obtained with monoclonal antibodies conjugated to ricin toxin Achain.

[0008] Due to the sensitivity, specificity, and adverse-effect profileof monoclonal antibodies, the results obtained using monoclonalantibody-based therapeutics have shown them to be less than idealtargeting tools. Although monoclonal antibodies have been generated toantigens selectively expressed on tumors, no truly cancer-specificantibody has been identified. Most antigens expressed on neoplasticcells appear to be quantitatively increased in these compared to normalcells but the antigens are nonetheless often present in normal cells.Thus, antibodies to such determinants can react with non-neoplastictissues, resulting in significant toxicities. Also, antibodies arerelatively large molecules and consequently, often evoke an immuneresponse in patients. These immune responses can result in significanttoxicities in patients upon re-exposure to the targeting agents and canprevent targeting by the monoclonal due to immune complex formation withdegradation and excretion. Finally, binding of antibodies to tumor cellsmay be low and targeted agents may be delivered to cells in quantitiesinsufficient to achieve detection or cytotoxicity.

[0009] There remains a need for reagents, kits and methods forscreening, diagnosing and monitoring individuals with colorectal cancer,particulalry metastasized colorectal cancer. There is a need forreagents, kits and methods for identifying and confirming that a cancerof unknown origin is colorectal cancer and for analyzing colon tissueand colorectal cancer samples to identify and confirm colorectal cancerand to determine the level of migration of such cancer cells. Thereremains a need for compositions which can specifically targetmetastasized colorectal cancer cells. There is a need for imaging agentswhich can specifically bind to metastasized colorectal cancer cells.There is a need for improved methods of imaging metastasized colorectalcancer cells. There is a need for therapeutic agents which canspecifically bind to metastasized colorectal cancer cells. There is aneed for improved methods of treating individuals who are suspected ofsuffering from colorectal cancer cells, especially individuals who aresuspected of suffering from metastasis of colorectal cancer cells. Thereis a need for vaccine composition to treat and prevent metastasizedcolorectal cancer. There is a need for therapeutic agents which canspecifically deliver gene therapeutics, antisense compounds and otherdrugs to colorectal cells.

SUMMARY OF THE INVENTION

[0010] The invention relates to isolated nucleic acid molecules thatcomprise a CRCA-1 transcript.

[0011] The invention relates to isolated nucleic acid molecules thatcomprise nucleic acid sequences that encode a substantially pure CRCA-1translation product or a functional fragment thereof. The inventionrelates to isolated nucleic acid molecules that comprise nucleic acidsequences that encode a substantially pure proteins that have amino acidsequences shown in SEQ ID NO:2-81.

[0012] The invention relates to isolated nucleic acid molecules thatcomprise nucleic acid sequences shown in SEQ ID NO:1 or a functionalfragment thereof.

[0013] The invention relates to pharmaceutical compositions thatcomprise nucleic acid molecule that comprise a CRCA-1 transcript.

[0014] The invention relates to pharmaceutical compositions thatcomprise nucleic acid molecule that comprise nucleic acid sequence shownin SEQ ID NO:1 or a functional fragment thereof in combination with apharmaceutically acceptable carrier.

[0015] The invention relates to a recombinant expression vectorcomprising the nucleic acid molecule that has a nucleotide sequence thatcomprises SEQ ID NO:1 or a functional fragment thereof.

[0016] The invention relates to a host cell comprising a recombinantexpression vector comprising the nucleic acid molecule that has anucleotide sequence that comprises SEQ ID NO:1 or a functional fragmentthereof.

[0017] The invention relates to an oligonucleotide molecule comprising anucleotide sequence complimentary to a nucleotide sequence of SEQ IDNO:1 or a functional fragment thereof.

[0018] The invention relates to isolated antibodies that bind to anepitope on a CRCA-1 translation product.

[0019] The invention relates to a substantially pure CRCA-1 translationproduct or a functional fragment thereof.

[0020] The invention relates to a substantially pure proteins that haveamino acid sequences shown in SEQ ID NO:2-81.

[0021] The invention relates to pharmaceutical compositions comprising asubstantially pure CRCA-1 translation product or a functional fragmentthereof.

[0022] The invention relates to pharmaceutical compositions comprising asubstantially pure protein having amino acid sequences shown in SEQ IDNO:2-81 in combination with a pharmaceutically acceptable carrier.

[0023] The invention relates to isolated antibodies that bind to anepitope on a protien having the amini acid sequence of SEQ ID NO:2-81.

[0024] The present invention relates to in vitro methods of determiningwhether or not an individual has metastasized colorectal cancer cells.The present invention relates to in vitro methods of examining samplesof non-colorectal tissue and body fluids from an individual to determinewhether or not a colorectal cancer specific transcript or a translationproduct thereof. CRC-1, which is specific to colorectal cells includingcolorectal tumor cells, is being expressed by cells outside of thecolorectal tract. The presence of a CRCA-1 translation product or of theCRCA-1 transcript outside the intestinal tract is indicative ofexpression of CRCA-1 and is evidence that the individual is sufferingfrom metastasized colorectal cancer.

[0025] The present invention relates to in vitro methods of determiningwhether or not tumor cells are colorectal in origin. The presentinvention relates to in vitro methods of diagnosing whether or not anindividual suffering from cancer is suffering from colorectal cancer.The present invention relates to in vitro methods of examining samplesof tumors from an individual to determine whether or not CRCA-1, whichis specific to colorectal cells including colorectal tumor cells, isbeing expressed by the tumor cells. The presence of a CRCA-1 translationproduct or of the CRCA-1 transcript is indicative of expression ofCRCA-1 is evidence that the individual is suffering from colorectalcancer.

[0026] The present invention relates to in vitro kits for practicing themethods of the invention and to reagents and compositions useful ascomponents in such in vitro kits of the invention.

[0027] The present invention relates to conjugated compounds whichcomprises a CRCA-1 translation product binding moiety and a radiostableactive moiety.

[0028] The present invention relates to a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent, and aconjugated compound which comprises a a CRCA-1 translation productbinding moiety and a radiostable active moiety.

[0029] The present invention relates to a method of treating anindividual suspected of suffering from metastasized colorectal cancercomprising the steps of administering to said individual apharmaceutical composition comprising a pharmaceutically acceptablecarrier or diluent, and a therapeutically effective amount of aconjugated compound which comprises a CRCA-1 translation product bindingmoiety and a radiostable active moiety.

[0030] The present invention relates to a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent, and aconjugated compound which comprises a a CRCA-1 translation productbinding moiety and a radioactive active moiety.

[0031] The present invention relates to a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent, andconjugated compound that comprises a CRCA-1 translation product bindingmoiety and a radioactive active moiety wherein the conjugated compoundis present in an amount effective for therapeutic or diagnostic use inhumans suffering from colorectal cancer.

[0032] The present invention relates to a method of radioimagingmetastasized colorectal cancer cells comprising the steps of firstadministering to an individual suspected of having metastasizedcolorectal cancer cells, a pharmaceutical composition that comprises apharmaceutically acceptable carrier or diluent, and conjugated compoundthat comprises a CRCA-1 translation product binding moiety and aradioactive active moiety wherein the conjugated compound is present inan amount effective for diagnostic use in humans suffering fromcolorectal cancer and then detecting the localization and accumulationof radioactivity in the individual's body.

[0033] The present invention relates to a method of treating anindividual suspected of suffering from metastasized colorectal cancercomprising the steps of administering to said individual apharmaceutical composition comprising a pharmaceutically acceptablecarrier or diluent, and a therapeutically effective amount of aconjugated compound which comprises a CRCA-1 translation product bindingmoiety and a radioactive active moiety.

[0034] The present invention relates to conjugated compounds whichcomprises a CRCA-1 translation product binding moiety and a activemoiety which comprises an antisense molecule.

[0035] The present invention relates to a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent, and aconjugated compound which comprises a CRCA-1 translation product bindingmoiety and an active moiety which comprises an antisense molecule.

[0036] The present invention relates to a method of treating anindividual suspected of suffering from colorectal cancer comprising thesteps of administering to a such an individual, a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand a therapeutically effective amount of a conjugated compound whichcomprises a CRCA-1 translation product binding moiety and an activemoiety which comprises an antisense molecule.

[0037] The present invention relates to a method of preventingcolorectal cancer in an individual comprising the steps of administeringto said individual a pharmaceutical composition comprising apharmaceutically acceptable carrier or diluent and a prophylacticallyeffective amount of a conjugated compound which comprises a CRCA-1translation product binding moiety and an active moiety which comprisesan antisense molecule.

[0038] The present invention relates to unconjugated compositionscomprises a liposome that includes CRCA-1 translation product ligands onits surface and an active component encapsualted therein.

[0039] The present invention relates to a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent, and anunconjugated compositions comprises a liposome that includes CRCA-1translation product ligands on its surface and an active componentencapsualted therein.

[0040] The present invention relates to a method of treating anindividual suspected of suffering from a disease, condition or disorderof the colon or of preventing such a disease, disorder or condition inan individual who is at risk of developing the same, comprising thesteps of administering to the colon a such an individual, apharmaceutical composition comprising a pharmaceutically acceptablecarrier or diluent, and an unconjugated compositions that comprises aliposome that includes CRCA-1 translation product ligands on its surfaceand an active component encapsualted therein.

[0041] The present invention relates to a method of delivering an activeagent to colon cells of an individual, including metastasized colorectalcancer cells comprising the steps of administering to such anindividual, a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or diluent, and an unconjugated compositions thatcomprises a liposome that includes CRCA-1 translation product ligands onits surface and an active component encapsualted therein.

[0042] The present invention relates to compositions that compriseCRCA-1 translation product ligands in combination with an activecomponent.

[0043] The present invention relates to a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent and acomposition that comprise CRCA-1 translation product ligands incombination with an active component.

[0044] The present invention relates to a method of treating anindividual suspected of suffering from a disease, condition or disorderof the colon or of preventing such a disease, disorder or condition inan individual who is at risk of developing the same, comprising thesteps of administering to the colon a such an individual, apharmaceutically acceptable carrier or diluent and a composition thatcomprise CRCA-1 translation product ligands in combination with anactive component.

[0045] The present invention relates to a method of delivering an activeagent to colon cells of an individual, including metastasized colorectalcancer cells comprising the steps of administering to such anindividual, a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or diluent and a composition that comprise CRCA-1translation product ligands in combination with an active component.

[0046] The present invention relates to an isolated protein comprisingat least one epitope of a CRCA-1 translation product. In someembodiments, the isolated protein comprises a CRCA-1 translationproduct. In some embodiments, the isolated protein consists of a CRCA-1translation product.

[0047] The present invention relates to vaccines which comprise suchproteins and a pharmaceutically acceptable carrier or diluent.

[0048] The present invention relates to a haptenized protein comprisingat least one epitope of a CRCA-1 translation product. In someembodiments, the haptenized protein comprises a CRCA-1 translationproduct. In some embodiments, the haptenized protein consists of aCRCA-1 translation product.

[0049] The present invention relates to vaccines which comprise suchhaptenized proteins and a pharmaceutically acceptable carrier ordiluent.

[0050] The present invention relates to nucleic acid molecules thatencode a protein comprising at least one epitope of a CRCA-1 translationproduct. In some embodiments, the nucleic acid molecule encodes aprotein that protein comprises a CRCA-1 translation product. In someembodiments, the nucleic acid molecule encodes a protein that consistsof a CRCA-1 translation product. In some embodiments, the nucleic acidmolecule encodes the CRCA-1 transcript. In some embodiments, the nucleicacid molecule is a plasmid.

[0051] The present invention relates to vaccines which comprise suchnucleic acid molecules and a pharmaceutically acceptable carrier ordiluent.

[0052] The present invention relates to vectors that comprise nucleicacid molecules that encode a protein comprising at least one epitope ofa CRCA-1 translation product. In some embodiments, the vector comprisesa nucleic acid molecule that encodes a protein that protein comprises aCRCA-1 translation product. In some embodiments, the vector comprises anucleic acid molecule that encodes a protein that consists of a CRCA-1translation product. In some embodiments, the vector comprises a nucleicacid molecule that is the CRCA-1 transcript. In some embodiments, thevector is a virus or a bacterial cell. In some embodiments, the vectoris a recombinant vaccinia virus.

[0053] The present invention relates to vaccines which comprise suchvectors and a pharmaceutically acceptable carrier or diluent.

[0054] The present invention relates to killed or inactivated cells orparticles that comprise a protein comprising at least one epitope of aCRCA-1 translation product. In some embodiments, the killed orinactivated cells or particles vector comprise the transmembrane domainof the human ST receptor protein. In some embodiments, the killed orinactivated cells or particles comprise a CRCA-1 translation product. Insome embodiments, the killed or inactivated cells or particles consistof a CRCA-1 translation product. In some embodiments, the killed orinactivated cells or particles vector is a killed or inactivatedcolorectal tumor cells.

[0055] The present invention relates to vaccines which comprise suchkilled or inactivated cells or particles and a pharmaceuticallyacceptable carrier or diluent.

[0056] The present invention relates to haptenized killed or inactivatedcells or particles that comprise a protein comprising at least oneepitope of a CRCA-1 translation product. In some embodiments, thehaptenized killed or inactivated cells or particles comprise a CRCA-1translation product. In some embodiments, the haptenized killed orinactivated cells or particles consist of a CRCA-1 translation product.In some embodiments, the haptenized killed or inactivated cells orparticles vector is a killed or inactivated colorectal tumor cells.

[0057] The present invention relates to vaccines which comprise suchhaptenized killed or inactivated cells or particles and apharmaceutically acceptable carrier or diluent.

[0058] The present invention relates to methods of treating individualssuffering from metastasized colorectal cancer. The method of the presentinvention provides administering to such an individual a therapeuticallyeffective amount of a vaccine of the invention. The invention relates tothe use of such vaccines as immunotherapeutics.

[0059] The present invention relates to methods of treating individualssusceptible metastasized colorectal cancer. The method of the presentinvention provides administering to such an individual amount of avaccine of the invention effective to prevent or combat metastasizedcolorectal cancer. The present invention relates to the use of thevaccines of the invention prophylactically.

BRIEF DESCRIPTION OF THE FIGURE

[0060]FIG. 1 shows the nucleotide sequence of the human ST receptormRNA, GeneBank Accession #S57551, which is incorporarted herein byreference. The gray shaded area is the sequence deleted in CRCA-1trascript including on fo the two boxed “GG” sequences or one G fromeach box. The start codon, ATG, which is nucleotides 118-119-120 of thesequence is the intiation codon for ST receptor protein expression. TheCRCA-1 transcript is missing a 142 nucleotide sequence spanningnucleotides 192-333, 193-334 or 194-335. Thus unique sequences of theCRCA-1 protein not found ST receptor mRNA include nucleotides191-192-193-336, 191-192-335-336 or 191-334-335-336 as set forth in FIG.1 or using corresponding numbers from sequences set forth in thesequence listing. These 4 nucleotide sequences are all identical,A-G-G-C, and correspond to nucleotides 110-111-112-113 of SEQ ID NO:1.

DETAILED DESCRIPTION OF THE INVENTION

[0061] Definitions

[0062] As used herein, the terms “ST” and “native ST” are usedinterchangeably and are meant to refer to heat-stable toxin (ST) whichis a peptide produced by E. coli, as well as other organisms. STs arenaturally occurring peptides which 1) are naturally produced byorganisms, 2) bind to the ST receptor and 3) activate the signal cascadethat mediates ST-induced diarrhea.

[0063] As used herein, the terms “ST receptor”, “guanylyl cyclase Creceptor” and “GCC receptor” are meant to refer to the receptors foundon colorectal cells, including local and metastasized colorectal cancercells, which bind to ST. In normal individuals, ST receptors are foundexclusively in cells of intestine, in particular in cells in theduodenum, small intestine (jejunum and ileum), the large intestine,colon (cecum, ascending colon, transverse colon, descending colon andsigmoid colon) and rectum.

[0064] As used herein, the terms “colorectal cancer-associatedtranscript” and “CRCA-1 transcript” are meant to refer to an alternativeform of the mRNA for the ST receptor produced by transcription of thehuman ST receptor gene. The CRCA-1 transcript possesses an alternativesequence from that of the ST receptor encoding-mRNA. CRCA-1 transcriptpreferably has a nucleotide sequence set forth in SEQ ID NO:1. CRCA-1transcript is found in colorectal cells, including local andmetastasized colorectal cancer cells. In normal individuals, CRCA-1transcript have been found exclusively in cells of intestine, inparticular in cells in the duodenum, small intestine (jejunum andileum), the large intestine, colon (cecum, ascending colon, transversecolon, descending colon and sigmoid colon) and rectum.

[0065] As used herein, the term “functional fragment” as used in theterm “functional fragment of a CRCA-1 transcript product” is meant tofragments of CRCA-1 transcript which are functional with respect tonucleic acid molecules with full length sequences. For example, afunctional fragment may be useful as an oligonucleotide or nucleic acidprobe, a primer, an antisense oligonucleotide or nucleic acid moleculeor a coding sequence. Functional fragments of the CRCA-1 transcript areunique compared to other known nucleic acid molecules, in particularfunctional fragments of the CRCA-1 transcript are unique compared tonucleic acid sequence of the ST receptor mRNA. The nucleotide sequenceencoding human ST receptor protein is disclosed in FIG. 1, SEQ ID NO:82and F. J. Sauvage et al. 1991 J. Biol. Chem. 266:17912-17918 which areincorporated herein by reference. The deleted sequence which results inthe generation fot he CRCA-1 transcript is disclosed in FIG. 1. Thus,the functional fragments of the CRCA-1 include specific sequences notfound on the ST recpetor mRNA. Such unique sequences include thesequences on either side of the deletion thus forming a unique sequencerelative to the ST receptor mRNA sequence. Accordingly, a functionalfragment will include nucleotides 110-113 of SEQ ID NO:1. It ispreferred that the unique sequence additionally include 5-10 or moresequences 5′ to nucleotide 110 and 5-10 or more sequences 3′ tonucleotide 113. Oligonucleotides and other fragments of the CRCA-1transcript which have sequences of function fragments includenucleitides 110-113 of SEQ ID NO:1 and ma additionally include sequences5′ and 3′ to the unique four nucleotide sequences formed by thedeletion. For example, a PCR primer having 8-28 nucleotides including aunique sequence for CRCA-1, i.e. a functional fragment having 8nucleotides may include nucleotide sequence 106-113 or 110-117 or an 8nucleotide seuquence generated from the intermediate sequences, i.e.107-114, 108-115 or 109-116, or a functional fragment having 28nucleotides may include nucleotide sequence 86-113 or 110-137 or a 28nucleotide sequence generated from the intermediate sequences.Similarly, other functional fragments of CRCA-1 transcript would include110-113 of SEQ ID NO:1 as part of a fragment of SEQ ID NO:1. Withrespect to CRCA-1 specific primers, sets of such primers may include oneunique fragment of CRCA-1 transcript and one primer which is notspecific for a unique CRCA-1 sequence provided that such a pair ofprimers can be used to amplify a CRCA-1 specific sequence.

[0066] As used herein, the terms “colorectal cancer-associatedtranslation products” and “CRCA-1 translation products” are meant torefer to translation products set forth in SEQ ID NOs:2-81. CRCA-1translation products are found in colorectal cells, including local andmetastasized colorectal cancer cells. In normal individuals, CRCA-1translation products have been found exclusively in cells of intestine,in particular in cells in the duodenum, small intestine (jejunum andileum), the large intestine, colon (cecum, ascending colon, transversecolon, descending colon and sigmoid colon) and rectum.

[0067] As used herein, the term “functional fragment” as used in theterm “functional fragment of a CRCA-1 translation product” is meant tofragments of CRCA-1 translation products which function in the samemanner as proteinaceous compounds with full length sequences. Forexample, an immunogenically functional fragment of a CRCA-1 comprises anepitope recognized by an anti-CRCA-1 translation product antibody. Aligand-binding functional fragment of a CRCA-1 comprises a sequencewhich forms a structure that can bind to a ligand which recognizes andbinds to a CRCA-1 translation product.

[0068] As used herein, the term “epitope recognized by an anti-CRCA-1translation product antibody” refers those epitopes recognized by ananti-CRCA-1 translation product antibody which does not recognizeepitopes of non-CRCA-1 translation products, i.e. does not cross reactwith non-CRCA-1 proteins.

[0069] As used herein, the term “antibody” is meant to refer tocomplete, intact antibodies, and Fab fragments and F(ab)₂ fragmentsthereof. Complete, intact antibodies include monoclonal antibodies suchas murine monoclonal antibodies, chimeric antibodies and humanizedantibodies.

[0070] As used herein, the term “CRCA-1 translation product ligand” ismeant to refer to compounds which specifically bind to a CRCA-1translation product. Antibodies that bind to a CRCA-1 translationproduct are CRCA-1 translation product ligands. An CRCA-1 translationproduct ligand may be a protein, peptide or a non-peptide.

[0071] As used herein, the term “active agent” is meant to refer tocompounds that are therapeutic agents or imaging agents.

[0072] As used herein, the term “radiostable” is meant to refer tocompounds which do not undergo radioactive decay; i.e. compounds whichare not radioactive.

[0073] As used herein, the term “therapeutic agent” is meant to refer tochemotherapeutics, toxins, radiotherapeutics, targeting agents orradiosensitizing agents.

[0074] As used herein, the term “chemotherapeutic” is meant to refer tocompounds that, when contacted with and/or incorporated into a cell,produce an effect on the cell including causing the death of the cell,inhibiting cell division or inducing differentiation.

[0075] As used herein, the term “toxin” is meant to refer to compoundsthat, when contacted with and/or incorporated into a cell, produce thedeath of the cell.

[0076] As used herein, the term “radiotherapeutic” is meant to refer toradionuclides which when contacted with and/or incorporated into a cell,produce the death of the cell.

[0077] As used herein, the term “targeting agent” is meant to refercompounds which can be bound by and or react with other compounds.Targeting agents may be used to deliver chemotherapeutics, toxins,enzymes, radiotherapeutics, antibodies or imaging agents to cells thathave targeting agents associated with them and/or to convert orotherwise transform or enhance co-administered active agents. Atargeting agent may include a moiety that constitutes a first agent thatis localized to the cell which when contacted with a second agent eitheris converted to a third agent which has a desired activity or causes theconversion of the second agent into an agent with a desired activity.The result is the localized agent facilitates exposure of an agent witha desired activity to the metastasized cell.

[0078] As used herein, the term “radiosensitizing agent” is meant torefer to agents which increase the susceptibility of cells to thedamaging effects of ionizing radiation. A radiosensitizing agent permitslower doses of radiation to be administered and still provide atherapeutically effective dose.

[0079] As used herein, the term “imaging agent” is meant to refer tocompounds which can be detected.

[0080] As used herein, the term “CRCA-1 translation product bindingmoiety” is meant to refer to the portion of a conjugated compound thatconstitutes an CRCA-1 translation product ligand.

[0081] As used herein, the term “active moiety” is meant to refer to theportion of a conjugated compound that constitutes an active agent.

[0082] As used herein, the terms “conjugated compound” and “conjugatedcomposition” are used interchangeably and meant to refer to a compoundwhich comprises an CRCA-1 translation product binding moiety and anactive moiety and which is capable of binding to the CRCA-1 translationproduct. it originally occurs to other parts of the body. The presentinvention relates to methods of delivering active agents to metastasizedcolorectal cancer cells.

[0083] As used herein, the term “metastasized colorectal cancer cells”is meant to refer to colorectal cancer cells which have metastasized;colorectal cancer cells localized in a part of the body other than theduodenum, small intestine (jejunum and ileum), large intestine (colon),including the cecum, ascending colon, transverse colon, descendingcolon, and sigmoid colon, and rectum.

[0084] As used herein, the term “non-colorectal sample” and“extra-intestinal sample” are used interchangeably and meant to refer toa sample of tissue or body fluid from a source other than colorectaltissue. In some preferred embodiments, the non-colorectal sample is asample of tissue such as lymph nodes. In some preferred embodiments, thenon-colorectal sample is a sample of extra-intestinal tissue which is anadenocarcinoma of unconfirmed origin. In some preferred embodiments, thenon-colorectal sample is a blood sample.

[0085] As used herein, “an individual suffering from an adenocarcinomaof unconfirmed origin” is meant to refer to an individual who has atumor in which the origin has not been definitively identified.

[0086] As used herein, “an individual is suspected of being susceptibleto metastasized colorectal cancer” is meant to refer to an individualwho is at a particular risk of developing metastasized colorectalcancer. Examples of individuals at a particular risk of developingmetastasized colorectal cancer are those whose family medical historyindicates above average incidence of colorectal cancer among familymembers and/or those who have already developed colorectal cancer andhave been effectively treated who therefore face a risk of relapse andrecurrence.

[0087] As used herein, the term “antisense composition” and “antisensemolecules” are used interchangeably and are meant to refer to compoundsthat regulate transcription or translation by hybridizing to DNA or RNAand inhibiting and/or preventing Conjugated compounds according to thepresent invention comprise a portion which constitutes an CRCA-1translation product ligand and a portion which constitutes an activeagent. Thus, conjugated compounds according to the present invention arecapable of specifically binding to the CRCA-1 translation product andinclude a portion which is a therapeutic agent or imaging agent.Conjugated compositions may comprise crosslinkers and/or molecules thatserve as spacers between the moieties.

[0088] As used herein, the terms “crosslinker”, “crosslinking agent”,“conjugating agent”, “coupling agent”, “condensation reagent” and“bifunctional crosslinker” are used interchangeably and are meant torefer to molecular groups which are used to attach the CRCA-1translation product ligand and the active agent to thus form theconjugated compound.

[0089] As used herein, the term “colorectal cancer” is meant to includethe well-accepted medical definition that defines colorectal cancer as amedical condition characterized by cancer of cells of the intestinaltract below the small intestine (i.e. the large intestine (colon),including the cecum, ascending colon, transverse colon, descendingcolon, and sigmoid colon, and rectum). Additionally, as used herein, theterm “colorectal cancer” is meant to further include medical conditionswhich are characterized by cancer of cells of the duodenum and smallintestine (jejunum and ileum). The definition of colorectal cancer usedherein is more expansive than the common medical definition but isprovided as such since the cells of the duodenum and small intestinealso contain CRCA-1 translation products and are therefore amenable tothe methods of the present invention using the compounds of the presentinvention.

[0090] As used herein, the term “metastasis” is meant to refer to theprocess in which cancer cells originating in one organ or part of thebody relocate to another part of the body and continue to replicate.Metastasized cells subsequently form tumors which may furthermetastasize. Metastasis thus refers to the spread of cancer from thepart of the body where transcription or translation from taking place.Antisense molecules include nucleic acid molecules and derivatives andanalogs thereof. Antisense molecules hybridize to DNA or RNA in the samemanner as complementary nucleotide sequences do regardless of whether ornot the antisense molecule is a nucleic acid molecule or a derivative oranalog. Antisense molecules inhibit or prevent transcription ortranslation of genes whose expression is linked to colorectal cancer.

[0091] As used herein, the term “CRCA-1 immunogen” is meant to refer toone or more CRCA-1 translation products or a fragment thereof or aprotein that comprises the same or a haptenized product thereof, cellsand particles which display at least one CRCA-1 epitope, and haptenizedcells and haptenized particles which display at least one CRCA-1epitope.

[0092] As used herein, the term “recombinant expression vector” is meantto refer to a plasmid, phage, viral particle or other vector which, whenintroduced into an appropriate host, contains the necessary geneticelements to direct expression of the coding sequence that encodes theprotein. The coding sequence is operably linked to the necessaryregulatory sequences. Expression vectors are well known and readilyavailable. Examples of expression vectors include plasmids, phages,viral vectors and other nucleic acid molecules or nucleic acid moleculecontaining vehicles useful to transform host cells and facilitateexpression of coding sequences.

[0093] ST Receptors and CRCA-1

[0094] Carcinomas derived from intestinal mucosa continue to express STreceptors on their cell surfaces. The expression of ST receptors bymetastatic tumors enables this protein and its mRNA to be a specificbiomarker for the presence of metastatic colorectal cancer cells inextra-intestinal tissues and blood. Indeed, this characteristic permitsthe detection of ST receptor mRNA by RT-PCR analysis to be a diagnostictest to stage patients with colorectal cancer and follow patients aftersurgery for evidence of recurrent disease in their blood. Further, theST receptor may be targeted with a ligand conjugated to an active agentin order to deliver the active agent to metastasized colotrectal tumorcells in vivo.

[0095] U.S. Pat. No. 5,518,888 issued May 21, 1996 to Waldman, PCTapplication PCT/US94/12232 filed Oct. 26, 1994, U.S. application Ser.No. 08/467,920 filed Jun. 6, 1995, and U.S. application Ser. No.08/583,447 filed Jan. 5, 1996, which are each incorporated herein byreference, disclose that metastized colorectal tumors can be targettedfor delivery of active compounds by targetting ST receptors. Thepresence of ST receptors on cells outside fo the intestinal tract as amarker for colorectal cancer allows for the screening, identificationand treatment of individuals with metastasized colorectal tumors. STreceptors may also be used to target delivery of gene therapeutics andantisense compounds to colorectal cells.

[0096] U.S. Pat. No. 5,601,990 issued Feb. 11, 1997, to Waldman, PCTapplication PCT/US94/12232 filed Oct. 26, 1994, and PCT applicationPCT/US97/07467 fileed May 2, 1997, which are each incorporated herein byreference, disclose that detection of evidence of expression of STreceptors in samples of tissue and body fluid from outside theintestinal tract indicate metastized colorectal cancer.

[0097] PCT application PCT/US97/07565 fileed May 2, 1997, which isincorporated herein by reference, disclose that immunogens with epitopesthat can be targetted by antibodies that react with ST receptors can beused in vaccines compositions useful as prophylactic and therapeuticanti-metastatic colorectal cancer compositions.

[0098] Recently, studies have identified an alternative form of the mRNAfor the ST receptor, isolated from human colon carcinoma cells. ThismRNA has a substantial deletion of nucleic acids in the first exon inthe coding region of the ST receptor. This deletion results in aframeshift of the coding region such that it no longer encodes the aminoacid sequence of the ST receptor. However, this alternative splicevariant mRNA appears to exhibit a selective pattern of expression thatparallels that of the ST receptor. This newly-identified mRNA has beendetected only in normal intestinal mucosal cells, human colorectaltumors, but not in extra-intestinal tissues. Furthermore, the expressionof this newly-identified mRNA can be detected by RT-PCR analysisseparately from ST receptor mRNA. Thus, the present invention providesthe use of this colorectal cancer-associated transcript (CRCA-1) as aspecific molecular diagnostic marker for the diagnosis, staging, andpost-operative surveillance of patients with colorectal cancer.

[0099] The newly-identified CRCA-1 appears to be a highly specificmarker for the diagnosis, staging, and post-operative surveillance ofpatients with colorectal cancer. Detection of the expression of CRCA-1employing molecular techniques, including, but not limited to, RT-PCR,can be employed to diagnose and stage patients, follow the developmentof recurrence after surgery, and, potentially, screen normal people forthe development of colorectal cancer. Detection of the expression ofCRCA-1 employing molecular techniques, including, but not limited to,RT-PCR, can be employed to diagnose and stage patients, follow thedevelopment of recurrence after surgery, and, potentially, screen normalpeople for the development of colorectal cancer.

[0100] The nucleotide sequence of the CRCA-1 transcription product isset forth as SEQ ID NO:1. Cells of colorectal origin may be distinguisedfrom cells of other origin based by detecting the presence or absence ofthe CRCA-1 transcription product.

[0101] It has further been discovered that one or more translationproducts may be produced from translation of the CRCA-1 transcriptionproduct. The transcription product contains a number of initiationcodons from which translation can begin, generating a number oftranslation products. Amino acid sequences of CRCA-1 translationproducts are set forth as SEQ. ID. No:2-81. Cells of colorectal originmay be distinguised from cells of other origin by detecting the presenceor absence of one or more of the CRCA-1 translation products.

[0102] ST receptors are unique in that they are only localized in theapical brush border membranes of the cells lining the intestinal tract.Indeed, they are not found in any other cell type in placental mammals.In addition, ST receptors are almost exclusively localized to the apicalmembranes, with little being found in the basolateral membranes on thesides of intestinal cells. Like ST receptors, the expression of CRCA-1is similarly localized.

[0103] Mucosal cells lining the intestine are joined together by tightjunctions which form a barrier against the passage of intestinalcontents into the blood stream and components of the blood stream intothe intestinal lumen. Therefore, the apical location of cells expressingST receptors and CRCA-1 isolates results in the isolation of such cellsfrom the circulatory system so that they may be considered to existseparate from the rest of the body; essentially the “outside” of thebody. Therefore, the rest of the body is considered “outside” theintestinal tract. Compositions administered “outside” the intestinaltract are maintained apart and segregated from the only cells whichnormally express ST receptors. Conversely, tissue samples taken fromtissue outside of the intestinal tract do not normally contain cellswhich express ST receptors and CRCA-1.

[0104] In individuals suffering from colorectal cancer, the cancer cellsare often derived from cells that produce and display the ST receptorand these cancer cells continue to produce and display the ST receptoron their cell surfaces. It has been observed that CRCA-1 is expressed bycolorectal cancer cells.

[0105] When such cancer cells metastasize, the metastasized cancer cellscontinue to produce and display the ST receptor. The expression ofCRCA-1 by metastatic tumor cells provides a detectable target for invitro screening, diagnosis, monitoring and staging as well as a targetfor in vivo delivery of conjugated compositions that comprise activeagents for the imaging and treatment.

[0106] The present invention relates to isolated CRCA-1 translationproducts. The present invention relates to isolated CRCA-1 transcript.The present invention relates to isolated antibodies specific for suchproducts and to hybridomas which produce such antibodies. Isolatedtranslation products or functional fragments thereof are useful togenrate antibodes according to the invention. Some aspects of theinvention nucleic acid molecules that encode the translation products.Nucleic acid molecules are useful to produce proteins which are be usedto generate antibodies.

[0107] In Vitro Diagnostics

[0108] According to some embodiments of the invention, compositions,kits and in vitro methods are provided for screening, diagnosiing andanalyzing patients and patient samples to detect evidence of CRCA-1expression by cells outside of the intestinal tract wherein theexpression of CRCA-1 is indicative of metastasis of colorectal cancer.Furthermore, the present invention relates to methods, compositions andkits useful in the in vitro screening, diagnosis and analysis of patientand patient samples to detect evidence of CRCA-1 expression by tumorcells outside of the intestinal tract wherein the presence of cells thatexpress CRCA-1 indicates and/or confirms that a tumor is of colorectalcancer origin. In an additional aspect of the invention, compositions,kits and methods are provided which are useful to visualize colorectalcells. Such compositions, kits and methods of analyzing tissue samplesfrom the colon tissue to evaluate the extent of metastasis or invasionof colorectal tumor cells into the lamina propria.

[0109] In vitro screening and diagnostic compositions, methods and kitscan be used in the monitoring of individuals who are in high risk groupsfor colorectal cancer such as those who have been diagnosed withlocalized disease and/or metastasized disease and/or those who aregenetically linked to the disease. In vitro screening and diagnosticcompositions, methods and kits can be used in the monitoring ofindividuals who are undergoing and/or have been treated for localizedcolorectal cancer to determine if the cancer has metastasized. In vitroscreening and diagnostic compositions, methods and kits can be used inthe monitoring of individuals who are undergoing and/or have beentreated for metastasized colorectal cancer to determine if themetastasized cancer has been eliminated. In vitro screening anddiagnostic compositions, methods and kits can be used in the monitoringof individuals who are otherwise susceptible, i.e. individuals who havebeen identified as genetically predisposed such as by genetic screeningand/or family histories. Advancements in the understanding of geneticsand developments in technology as well as epidemiology allow for thedetermination of probability and risk assessment an individual has fordeveloping colorectal cancer. Using family health histories and/orgenetic screening, it is possible to estimate the probability that aparticular individual has for developing certain types of cancerincluding colorectal cancer. Those individuals that have been identifiedas being predisposed to developing a particular form of cancer can bemonitored or screened to detect evidence of metastasized colorectalcancer. Upon discovery of such evidence, early treatment can beundertaken to combat the disease. Accordingly, individuals who are atrisk for developing metastasized colorectal cancer may be identified andsamples may be isolated form such individuals. The invention isparticularly useful for monitoring individuals who have been identifiedas having family medical histories which include relatives who havesuffered from colorectal cancer. Likewise, the invention is particularlyuseful to monitor individuals who have been diagnosed as havingcolorectal cancer and, particularly those who have been treated and hadtumors removed and/or are otherwise experiencing remission includingthose who have been treated for metastasized colorectal cancer.

[0110] In vitro screening and diagnostic compositions, methods and kitscan be used in the analysis of tumors. Expression of CRCA-1 is a markerfor cell type and allows for the identification of the origin ofadenocarcinoma of unconfirmed origin as colorectal tumors as well asallowing for an initial diagnosis of colorectal cancer to be confirmed.Tumors believed to be colorectal in origin can be confirmed as suchusing the compositions, methods and kits of the invention. The inventioncan be used to confirm the diagnosis of colorectal cancer by confirmingthat tumors are of colorectal origin. Similarly, tumors of unknownorigin can be analyzed and identified as being colorectal in originusing the compositions, methods and kits of the invention. The inventioncan be used to identify colorectal tumors in samples of tumors removedfrom individuals suffering from adenocarcinomas of unconfirmed origin.

[0111] In vitro screening and diagnostic compositions, kits and methodsof the invention can be used to analyze tissue samples from the colontissue to evaluate the extent of metastasis or invasion of colorectaltumor cells into the lamina propria. The lamina propria represents thebarrier between the colorectal tract and the rest of the body; seeBailey's Textbook of Histology, 16th edition, Coperhaven et al. 1975Williams and Wilkens, Baltimore Md. at page 404 which is incorporatedherein by reference. By identifying the presence of CRCA-1 transcript ortranslation products in cells of the lamina propria, the extent ofinvasion/infiltration of colorectal tumor cells into non-colorectaltissue can be evaluated and confirmed.

[0112] According to the invention, compounds are provided which bind toCRCA-1 transcript or translation products. Normal tissue in the bodydoes not have CRCA-1 transcript or translation products except cells ofthe intestinal tract. Metastasized colorectal cells may be identified bydetecting in non-colorectal samples CRCA-1 transcript or translationproducts. The expression of CRCA-1 is a marker for cell type and allowsfor the identification of colorectal metastasis in extra-intestinalsamples. CRCA-1 transcript or translation products may be used tovisualize colorectal derived cells from other cells of the lumen inorder to evaluate the level of invasion of colorectal tumor cells intothe basement membrane.

[0113] In some embodiments of the invention, non-colorectal tissue andfluid samples or tumor samples may be screened to identify the presenceor absence of CRCA-1 translation products. Techniques such as ELISAassays and Western blots may be performed to determine whether one ormore CRCA-1 translation products are present in a sample.

[0114] In some embodiments of the invention, non-colorectal tissue andfluid samples or tumor samples may be screened to identify whether oneor more CRCA-1 translation products are being expressed in cells outsideof the colorectal tract by detecting the presence or absence of CRCA-1transcript. The presence of CRCA-1 transcript or cDNA generatedtherefrom can be determined using techniques such as PCR amplification,branched oligonucleotide technology, Northern Blots (mRNA), SouthernBlots (cDNA), or oligonucleotide hybridization.

[0115] In some embodiments of the invention, cells of non-colorectaltissue samples or tumor samples may be examined to identify the presenceor absence of one or more CRCA-1 translation products. Techniques suchas immunohistochemistry blots may be performed on tissue sections todetermine whether one or more CRCA-1 translation products are present ina sample.

[0116] In some embodiments of the invention, cells of non-colorectaltissue samples or tumor samples may be examined to determine whether oneor more CRCA-1 translation products is being expressed in cells outsideof the colorectal tract by detecting the presence or absence of theCRCA-1 transcript. The presence of the CRCA-1 transcript or cDNAgenerated therefrom in cells from tissue sections can be determinedusing techniques such as in situ hybridization.

[0117] The presence of one or more CRCA-1 translation products innon-colorectal tissue and fluid samples or on cells from non-colorectaltissue samples indicates colorectal tumor metastasis. The presence ofone or more CRCA-1 translation products in a tumor sample or on tumorcells indicates that the tumor is colorectal in origin. The presence ofthe CRCA-1 transcript in non-colorectal tissue and fluid samples or incells from non-colorectal tissue samples indicates colorectal tumormetastasis. The presence of the CRCA-1 transcript in tumor samples andtumor cells indicates that the tumor is colorectal in origin.

[0118] Some aspects of the present invention relate to methods and kitsfor evaluating the metastatic migration of tumor cells in the laminapropria, indicating the level of invasion of colorectal tumor cells intothe basement membrane. In some embodiments of the invention, tissuesamples which include sections of the lamina propria may be isolatedfrom individuals undergoing or recovery from surgery to removecolorectal tumors. The tissue is analyzed to determine the extent ofinvasion into the basement membrane of the lamina propria by neoplasticcolorectal cells. Identification of the presence or absence of the oneor more CRCA-1 translation products confirms evaluation of the migrationof tumor cells into the basement membrane indicating metastasis.Techniques such as immunohistochemistry assays may be performed todetermine whether one or more CRCA-1 translation products are present incells in the tissue sample which are indicative of metastatic migration.Alternatively, in some embodiments of the invention, tissue samples thatinclude the lamina propria are analyzed to identify whether one or moreCRCA-1 translation products are being expressed in cells in the tissuesample which indicate metastatic migration by detecting the presence orabsence of the CRCA-1 transcript. The presence of the CRCA-1 transcriptor cDNA generated therefrom can be determined using techniques such asin situ hybridization.

[0119] Samples from tumors may be identified as colorectal in origin byidentification of expression of one or more CRCA-1 translation productsusing the methods of the invention. Samples of tumors removed fromindividuals suffering from adenocarcinomas of unconfirmed origin can betested to determine whether or not they possess one or more CRCA-1translation products or the CRCA-1 transcript. If the sample is removedfrom the intestinal tract, a section of frozen cells can be examined todetermine if the tumor cells express one or more CRCA-1 translationproducts. If the sample is removed from the extra-intestinal tissue, asection of frozen cells can be examined to determine if the tumor cellsexpress one or more CRCA-1 translation products or the sample can behomogenized and tested since the non-cancer cells will not possess oneor more CRCA-1 translation products and therefore not presentbackground.

[0120] Samples may be obtained from resected tissue or biopsy materialincluding needle biopsy. Tissue section preparation for surgicalpathology may be frozen and prepared using standard techniques.Immunohistochemistry and in situ hybridization binding assays on tissuesections are performed in fixed cells. Extra-intestinal samples may behomogenized by standard techniques such as sonication, mechanicaldisruption or chemical lysis such as detergent lysis. It is alsocontemplated that tumor samples in body such as blood, urine, lymphfluid, cerebral spinal fluid, amniotic fluid, vaginal fluid, semen andstool samples may also be screened to determine if such tumors arecolorectal in origin.

[0121] Non-colorectal tissue samples may be obtained from any tissueexcept those of the colorectal tract, i.e. the intestinal tract belowthe small intestine (i.e. the large intestine (colon), including thececum, ascending colon, transverse colon, descending colon, and sigmoidcolon, and rectum) and additionally the duodenum and small intestine(jejunum and ileum). The cells of all tissue except those of thecolorectal tract do not express one or more CRCA-1 translation products.Thus if one or more CRCA-1 translation products or the CRCA-1 transcriptare detected in non-colorectal samples, the presence of metastaticcolorectal cancer cells is indicated. In some preferred embodiments, thetissue samples are lymph nodes.

[0122] Tissue samples may be obtained by standard surgical techniquesincluding use of biopsy needles. One skilled in the art would readilyappreciate the variety of test samples that may be examined for one ormore CRCA-1 translation products and recognize methods of obtainingtissue samples.

[0123] Tissue samples may be homogenized or otherwise prepared forscreening for the presence of one or more CRCA-1 translation products bywell known techniques such as sonication, mechanical disruption,chemical lysis such as detergent lysis or combinations thereof.

[0124] Examples of body fluid samples include blood, urine, lymph fluid,cerebral spinal fluid, amniotic fluid, vaginal fluid and semen. In somepreferred embodiments, blood is used as a sample of body fluid. Cellsmay be isolated from fluid sample such as centrifugation. One skilled inthe art would readily appreciate the variety of test samples that may beexamined for one or more CRCA-1 translation products. Test samples maybe obtained by such methods as withdrawing fluid with a syringe or by aswab. One skilled in the art would readily recognize other methods ofobtaining test samples.

[0125] In an assay using a blood sample, the blood plasma may beseparated from the blood cells. The blood plasma may be screened for oneor more CRCA-1 translation products including truncated proteins whichare released into the blood when one or more CRCA-1 translation productsare cleaved from or sloughed off from metastasized colorectal tumorcells. In some embodiments, blood cell fractions are screened for thepresence of metastasized colorectal tumor cells. In some embodiments,lymphocytes present in the blood cell fraction are screened by lysingthe cells and detecting the presence of one or more CRCA-1 translationproducts or the CRCA-1 transcript which may be present as a result ofthe presence of any metastasized colorectal tumor cells that may havebeen engulfed by the blood cell.

[0126] For aspects of the invention related to analysis of lumen tissue,the invention is useful to evaluate the level of metastatic migration ofcolorectal tumor cells using lumen samples taken from surgery patientsat and near the site of the tumor. Some aspects of the invention providemethods of analyzing tissue samples which are fixed sections routinelyprepared by surgical pathologists to characterize and evaluate cells. Insome embodiments, the cells are from lamina propria and are analyzed todetermine and evaluate the extent of metastasis of colorectal tumorcells. The lamina propria represents the barrier between the colorectaltract and the rest of the body. By identifying the presence of theCRCA-1 transcript or one or more CRCA-1 translation products in cells ofthe lamina propria, the extent of invasion/infiltration of colorectaltumor cells into non-colorectal tissue can be evaluated. In someembodiments, the cells are removed in a biopsy or as an adenocarcinomaof unknown origin and are analyzed to determine and evaluate the whetherthey are colorectal tumor cells. In some embodiments, the cells are froma tumor suspected of being colorectal in origin and the method andcompositions and kits of the invention are used to confirm the identityof the origin of the tumor cells.

[0127] Samples of the lamina propria are removed during colorectal tumorremoval surgery such as by resection or colonoscopy. The sampleincluding basement membrane cells is frozen. If immunohistochemistry orin situ hybridization is to be performed, the frozen section is stainedand then the assay is run. Those having ordinary skill in the art canreadily isolate samples which include portions of the lamina propria andfix and stain them using standard techniques. By adding thevisualization provided with a CRCA-1 detection technique, the sectioncan be more comprehensively analyzed and the level of invasion ofneoplastic colorectal cells into the lamina propria can be determined.The present invention may be used to analyze and evaluate the extent ofprogression of localized colorectal tumors, that is primary ornon-metastatic colorectal tumors if these have penetrated the basementmembrane underlying the mucosa into the submucosa.

[0128] Immunoassay methods may be used to identify individuals sufferingfrom colorectal cancer metastasis by detecting presence of one or moreCRCA-1 translation products in sample of non-colorectal tissue or bodyfluid using antibodies which were produced in response to exposure tosuch CRCA-1 translation product. Moreover, immunoassay methods may beused to identify individuals suffering from colorectal cancer bydetecting presence of one or more CRCA-1 translation products in sampleof tumor using antibodies which were produced in response to exposure tosuch CRCA-1 translation product.

[0129] The antibodies are preferably monoclonal antibodies. Theantibodies are preferably raised against one or more CRCA-1 translationproducts made in human cells. Immunoassays are well known and theredesign may be routinely undertaken by those having ordinary skill in theart. Those having ordinary skill in the art can produce monoclonalantibodies which specifically bind to one of the several CRCA-1translation products and are useful in methods and kits of the inventionusing standard techniques and readily available starting materials. Thetechniques for producing monoclonal antibodies are outlined in Harlow,E. and D. Lane, (1988) ANTIBODIES: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor N.Y., which is incorporated hereinby reference, provide detailed guidance for the production of hybridomasand monoclonal antibodies which specifically bind to target proteins. Itis within the scope of the present invention to include FAbs and F(Ab)2swhich specifically bind to one or more CRCA-1 translation products inplace of antibodies.

[0130] Briefly, a CRCA-1 translation product is injected into mice. Thespleen of the mouse is removed, the spleen cells are isolated and fusedwith immortalized mouse cells. The hybrid cells, or hybridomas, arecultured and those cells which secrete antibodies are selected. Theantibodies are analyzed and, if found to specifically bind to the CRCA-1translation product, the hybridoma which produces them is cultured toproduce a continuous supply of anti-CRCA-1 translation product specificantibodies.

[0131] The present invention relates to antibodies which are produced inresponse to exposure to a CRCA-1 translation product. The antibodies arepreferably monoclonal antibodies. The antibodies are preferably raisedagainst CRCA-1 translation product made in human cells.

[0132] The means to detect the presence of a protein in a test sampleare routine and one having ordinary skill in the art can detect thepresence or absence of a protein or an antibody using well knownmethods. One well known method of detecting the presence of a protein isan immunoassay. One having ordinary skill in the art can readilyappreciate the multitude of ways to practice an immunoassay to detectthe presence of a CRCA-1 translation product in a sample.

[0133] According to some embodiments, immunoassays comprise allowingproteins in the sample to bind a solid phase support such as a plasticsurface. Detectable antibodies are then added which selectively bindingto either the CRCA-1 translation product. Detection of the detectableantibody indicates the presence of CRCA-1 translation product. Thedetectable antibody may be a labelled or an unlabelled antibody.Unlabelled antibody may be detected using a second, labelled antibodythat specifically binds to the first antibody or a second, unlabelledantibody which can be detected using labelled protein A, a protein thatcomplexes with antibodies. Various immunoassay procedures are describedin Immunoassays for the 80's, A. Voller et al., Eds., University Park,1981, which is incorporated herein by reference.

[0134] Simple immunoassays may be performed in which a solid phasesupport is contacted with the test sample. Any proteins present in thetest sample bind the solid phase support and can be detected by aspecific, detectable antibody preparation. Such a technique is theessence of the dot blot, Western blot and other such similar assays.

[0135] Other immunoassays may be more complicated but actually provideexcellent results. Typical and preferred immunometric assays include“forward” assays for the detection of a protein in which a firstanti-protein antibody bound to a solid phase support is contacted withthe test sample. After a suitable incubation period, the solid phasesupport is washed to remove unbound protein. A second, distinctanti-protein antibody is then added which is specific for a portion ofthe specific protein not recognized by the first antibody. The secondantibody is preferably detectable. After a second incubation period topermit the detectable antibody to complex with the specific proteinbound to the solid phase support through the first antibody, the solidphase support is washed a second time to remove the unbound detectableantibody. Alternatively, the second antibody may not be detectable. Inthis case, a third detectable antibody, which binds the second antibodyis added to the system. This type of “forward sandwich” assay may be asimple yes/no assay to determine whether binding has occurred or may bemade quantitative by comparing the amount of detectable antibody withthat obtained in a control. Such “two-site” or “sandwich” assays aredescribed by Wide, Radioimmune Assay Method, Kirkham, Ed., E. & S.Livingstone, Edinburgh, 1970, pp. 199-206, which is incorporated hereinby reference.

[0136] Other types of immunometric assays are the so-called“simultaneous” and “reverse” assays. A simultaneous assay involves asingle incubation step wherein the first antibody bound to the solidphase support, the second, detectable antibody and the test sample areadded at the same time. After the incubation is completed, the solidphase support is washed to remove unbound proteins. The presence ofdetectable antibody associated with the solid support is then determinedas it would be in a conventional “forward sandwich” assay. Thesimultaneous assay may also be adapted in a similar manner for thedetection of antibodies in a test sample.

[0137] The “reverse” assay comprises the stepwise addition of a solutionof detectable antibody to the test sample followed by an incubationperiod and the addition of antibody bound to a solid phase support afteran additional incubation period. The solid phase support is washed inconventional fashion, to remove unbound protein antibody complexes andunreacted detectable antibody. The determination of detectable antibodyassociated with the solid phase support is then determined as in the“simultaneous” and “forward” assays. The reverse assay may also beadapted in a similar manner for the detection of antibodies in a testsample.

[0138] The first component of the immunometric assay may be added tonitrocellulose or other solid phase support which is capable ofimmobilizing proteins. The first component for determining the presenceof CRCA-1 translation product in a test sample is an anti-CRCA-1translation product antibody. By “solid phase support” or “support” isintended any material capable of binding proteins. Well-known solidphase supports include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the support canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support configuration may be spherical, as in abead, or cylindrical, as in the inside surface of a test tube or theexternal surface of a rod. Alternatively, the surface may be flat suchas a sheet, test strip, etc. Those skilled in the art will know manyother suitable “solid phase supports” for binding proteins or will beable to ascertain the same by use of routine experimentation. Apreferred solid phase support is a 96-well microtiter plate.

[0139] To detect the presence of one or more CRCA-1 translationproducts, detectable anti-CRCA-1 translation product antibodies areused. Several methods are well known for the detection of antibodies.

[0140] One method in which the antibodies can be detectably labelled isby linking the antibodies to an enzyme and subsequently using theantibodies in an enzyme immunoassay (EIA) or enzyme-linked immunosorbentassay (ELISA), such as a capture ELISA. The enzyme, when subsequentlyexposed to its substrate, reacts with the substrate and generates achemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Enzymes which can beused to detectably label antibodies include, but are not limited tomalate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. One skilled in the art wouldreadily recognize other enzymes which may also be used.

[0141] Another method in which antibodies can be detectably labelled isthrough radioactive isotopes and subsequent use in a radioimmunoassay(RIA) (see, for example, Work, T. S. et al., Laboratory Techniques andBiochemistry in Molecular Biology, North Holland Publishing Company,N.Y., 1978, which is incorporated herein by reference). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography. Isotopes which areparticularly useful for the purpose of the present invention are ³H,¹²⁵I, ¹³¹I, ³⁵S, and ¹⁴C. Preferably ¹²⁵I is the isotope. One skilled inthe art would readily recognize other radioisotopes which may also beused.

[0142] It is also possible to label the antibody with a fluorescentcompound. When the fluorescent-labelled antibody is exposed to light ofthe proper wave length, its presence can be detected due to itsfluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Oneskilled in the art would readily recognize other fluorescent compoundswhich may also be used.

[0143] Antibodies can also be detectably labelled usingfluorescence-emitting metals such as ¹⁵²Eu, or others of the lanthanideseries. These metals can be attached to the protein-specific antibodyusing such metal chelating groups as diethylenetriaminepentaacetic acid(DTPA) or ethylenediamine-tetraacetic acid (EDTA). One skilled in theart would readily recognize other fluorescence-emitting metals as wellas other metal chelating groups which may also be used.

[0144] Antibody can also be detectably labelled by coupling to achemiluminescent compound. The presence of the chemiluminescent-labelledantibody is determined by detecting the presence of luminescence thatarises during the course of a chemical reaction. Examples ofparticularly useful chemoluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. One skilled in the art would readily recognize otherchemiluminescent compounds which may also be used. Likewise, abioluminescent compound may be used to label antibodies. Bioluminescenceis a type of chemiluminescence found in biological systems in which acatalytic protein increases the efficiency of the chemiluminescentreaction. The presence of a bioluminescent protein is determined bydetecting the presence of luminescence. Important bioluminescentcompounds for purposes of labeling are luciferin, luciferase andaequorin. One skilled in the art would readily recognize otherbioluminescent compounds which may also be used.

[0145] Detection of the protein-specific antibody, fragment orderivative may be accomplished by a scintillation counter if, forexample, the detectable label is a radioactive gamma emitter.Alternatively, detection may be accomplished by a fluorometer if, forexample, the label is a fluorescent material. In the case of an enzymelabel, the detection can be accomplished by colorometric methods whichemploy a substrate for the enzyme. Detection may also be accomplished byvisual comparison of the extent of enzymatic reaction of a substrate incomparison with similarly prepared standards. One skilled in the artwould readily recognize other appropriate methods of detection which mayalso be used.

[0146] The binding activity of a given lot of antibodies may bedetermined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

[0147] Positive and negative controls may be performed in which knownamounts of one or more CRCA-1 translation products and no CRCA-1translation product, respectively, are added to assays being performedin parallel with the test assay. One skilled in the art would have thenecessary knowledge to perform the appropriate controls. In addition,the kit may comprise instructions for performing the assay. Additionallythe kit may optionally comprise depictions or photographs that representthe appearence of positive and negative results.

[0148] CRCA-1 translation products may be produced as a reagent forpositive controls routinely. One skilled in the art would appreciate thedifferent manners in which the CRCA-1 translation products may beproduced and isolated.

[0149] Antibody composition refers to the antibody or antibodiesrequired for the detection of the protein. For example, the antibodycomposition used for the detection of a CRCA-1 translation product in atest sample comprises a first antibody that binds to the CRCA-1translation product as well as a second or third detectable antibodythat binds the first or second antibody, respectively.

[0150] To examine a test sample for the presence of a CRCA-1 translationproduct, a standard immunometric assay such as the one described belowmay be performed. A first anti-CRCA-1 translation product antibody,which recognizes a specific portion of CRCA-1 translation product, isadded to a 96-well microtiter plate in a volume of buffer. The plate isincubated for a period of time sufficient for binding to occur andsubsequently washed with PBS to remove unbound antibody. The plate isthen blocked with a PBS/BSA solution to prevent sample proteins fromnon-specifically binding the microtiter plate. Test sample aresubsequently added to the wells and the plate is incubated for a periodof time sufficient for binding to occur. The wells are washed with PBSto remove unbound protein. Labelled anti-CRCA-1 translation productantibodies, which recognize portions of CRCA-1 translation product notrecognized by the first antibody, are added to the wells. The plate isincubated for a period of time sufficient for binding to occur andsubsequently washed with PBS to remove unbound, labelled anti-CRCA-1translation product antibody. The amount of labelled and boundanti-CRCA-1 translation product antibody is subsequently determined bystandard techniques.

[0151] Kits which are useful for the detection of a CRCA-1 translationproduct in a test sample comprise a container comprising anti-CRCA-1translation product antibodies and a container or containers comprisingcontrols. Controls include one control sample which does not containCRCA-1 translation product and/or another control sample which containedthe CRCA-1 translation product. The anti-CRCA-1 translation productantibodies used in the kit are detectable such as being detectablylabelled. If the detectable anti-CRCA-1 translation product antibody isnot labelled, it may be detected by second antibodies or protein A forexample which may also be provided in some kits in separate containers.Additional components in some kits include solid support, buffer, andinstructions for carrying out the assay. Additionally the kit mayoptionally comprise depictions or photographs that represent theappearence of positive and negative results.

[0152] The immunoassay is useful for detecting one or more CRCA-1translation products in homogenized tissue samples and body fluidsamples including the plasma portion or cells in the fluid sample.

[0153] Western Blots may be used in methods of identifying individualssuffering from colorectal cancer metastasis by detecting presence of oneor more CRCA-1 translation products of non-colorectal tissue or bodyfluid. Western blots may also be used to detect presence of one or moreCRCA-1 translation products in sample of tumor from an individualsuffering from cancer to identify and/or confirm that the tumor iscolorectal in origin. Western blots use detectable anti-CRCA-1translation product antibodies to bind to any CRCA-1 translation productpresent in a sample and thus indicate the presence of the receptor inthe sample.

[0154] Western blot techniques, which are described in Sambrook, J. etal., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., which is incorporated hereinby reference, are similar to immunoassays with the essential differencebeing that prior to exposing the sample to the antibodies, the proteinsin the samples are separated by gel electrophoresis and the separatedproteins are then probed with antibodies. In some preferred embodiments,the matrix is an SDS-PAGE gel matrix and the separated proteins in thematrix are transferred to a carrier such as filter paper prior toprobing with antibodies. Anti-CRCA-1 translation product antibodiesdescribed above are useful in Western blot methods.

[0155] Generally, samples are homogenized and cells are lysed usingdetergent such as Triton-X. The material is then separated by thestandard techniques in Sambrook, J. et al., (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

[0156] Kits which are useful for the detection of one or more CRCA-1translation products in a test sample by Western Blot comprise acontainer comprising anti-CRCA-1 translation products antibodies and acontainer or containers comprising controls. Controls include onecontrol sample which does not contain CRCA-1 translation product and/oranother control sample which contained one or more CRCA-1 translationproducts. The anti-CRCA-1 translation product antibodies used in the kitare detectable such as being detectably labelled. If the detectableanti-CRCA-1 translation product antibody is not labelled, it may bedetected by second antibodies or protein A for example which may also beprovided in some kits in separate containers. Additional components insome kits include instructions for carrying out the assay. Additionallythe kit may optionally comprise depictions or photographs that representthe appearence of positive and negative results.

[0157] Western blots are useful for detecting one or more CRCA-1translation products in homogenized tissue samples and body fluidsamples including the plasma portion or cells in the fluid sample.

[0158] In addition to detection of one or more CRCA-1 translationproducts, aspects of the present invention include various methods ofdetermining whether a sample contains cells that express CRCA-1 bynucleotide sequence-based molecular analysis to detect the CRCA-1transcript. Several different methods are available for doing soincluding those using Polymerase Chain Reaction (PCR) technology,branched oligonucleotide technology, Northern blot technology,oligonucleotide hybridization technology, and in situ hybridizationtechnology.

[0159] The invention relates to oligonucleotide probes and primers usedin the methods of identifying the CRCA-1 transcript and to diagnostickits which comprise such components. The mRNA sequence-based methods fordetect the CRCA-1 transcript include but are not limited to polymerasechain reaction technology, branched oligonucleotide technology, Northernand Southern blot technology, in situ hybridization technology andoligonucleotide hybridization technology.

[0160] The methods described herein are meant to exemplify how thepresent invention may be practiced and are not meant to limit the scopeof invention. It is contemplated that other sequence-based methodologyfor detecting the presence of the CRCA-1 transcript in non-colorectalsamples may be employed according to the invention.

[0161] A preferred method to detecting the CRCA-1 transcript in geneticmaterial derived from non-colorectal samples uses polymerase chainreaction (PCR) technology. PCR technology is practiced routinely bythose having ordinary skill in the art and its uses in diagnostics arewell known and accepted. Methods for practicing PCR technology aredisclosed in “PCR Protocols: A Guide to Methods and Applications”,Innis, M. A., et al. Eds. Academic Press, Inc. San Diego, Calif. (1990)which is incorporated herein by reference. Applications of PCRtechnology are disclosed in “Polymerase Chain Reaction” Erlich, H. A.,et al., Eds. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)which is incorporated herein by reference. U.S. Pat. No. 4,683,202, U.S.Pat. No. 4,683,195, U.S. Pat. No. 4,965,188 and U.S. Pat. No. 5,075,216,which are each incorporated herein by reference describe methods ofperforming PCR. PCR may be routinely practiced using Perkin Elmer CetusGENE AMP RNA PCR kit, Part No. N808-0017.

[0162] PCR technology allows for the rapid generation of multiple copiesof DNA sequences by providing 5′ and 3′ primers that hybridize tosequences present in an RNA or DNA molecule, and further providing freenucleotides and an enzyme which fills in the complementary bases to thenucleotide sequence between the primers with the free nucleotides toproduce a complementary strand of DNA. The enzyme will fill in thecomplementary sequences adjacent to the primers. If both the 5′ primerand 3′ primer hybridize to nucleotide sequences on the same smallfragment of nucleic acid, exponential amplification of a specificdouble-stranded size product results. If only a single primer hybridizesto the nucleic acid fragment, linear amplification producessingle-stranded products of variable length.

[0163] PCR primers can be designed routinely by those having ordinaryskill in the art using sequence information. The nucleotide sequence ofthe the CRCA-1 transcript is set forth in SEQ ID NO:1. To perform thismethod, RNA is extracted from cells in a sample and tested or used tomake cDNA using well known methods and readily available startingmaterials. Those having ordinary skill in the art can readily preparePCR primers. A set of primers generally contains two primers. Whenperforming PCR on extracted mRNA or cDNA generated therefrom, if theCRCA-1 transcript or cDNA generated thererefrom is present, multiplecopies of the mRNA or cDNA will be made. If it is not present, PCR willnot generate a discrete detectable product. Primers are generally 8-50nucleotides, preferably about 15-35 nucleotides, more preferably 18-28nucleotides, which are identical or complementary to and thereforhybridize to the CRCA-1 transcript or cDNA generated therefrom. Inpreferred embodiments, the primers are each 15-35 nucleotide, morepreferably 18-28 nucleotide fragments of SEQ ID NO:1. The primer musthybridize to the sequence to be amplified. Typical primers are 18-28nucleotides in length and are generally have 50% to 60% G+C composition.The entire primer is preferably complementary to the sequence it musthybridize to. Preferably, primers generate PCR products 100 base pairsto 2000 base pairs. However, it is possible to generate products of 50to up to 10 kb and more. If mRNA is used as a template, the primers musthybridize to mRNA sequences. If cDNA is used as a template, the primersmust hybridize to cDNA sequences. At least one primer hybridizes to aunique nucleotide sequence not found on mRNA that encodes ST receptorprotein.

[0164] The mRNA or cDNA is combined with the primers, free nucleotidesand enzyme following standard PCR protocols. The mixture undergoes aseries of temperature changes. If the CRCA-1 transcript or cDNAgenerated therefrom is present, that is, if both primers hybridize tosequences on the same molecule, the molecule comprising the primers andthe intervening complementary sequences will be exponentially amplified.The amplified DNA can be easily detected by a variety of well knownmeans. If no CRCA-1 transcript or cDNA generated therefrom is present,no PCR product will be exponentially amplified. The PCR technologytherefore provides an extremely easy, straightforward and reliablemethod of detecting the CRCA-1 transcript in a sample.

[0165] PCR product may be detected by several well known means. Thepreferred method for detecting the presence of amplified DNA is toseparate the PCR reaction material by gel electrophoresis and stain thegel with ethidium bromide in order to visual the amplified DNA ifpresent. A size standard of the expected size of the amplified DNA ispreferably run on the gel as a control.

[0166] In some instances, such as when unusually small amounts of RNAare recovered and only small amounts of cDNA are generated therefrom, itis desirable or necessary to perform a PCR reaction on the first PCRreaction product. That is, if difficult to detect quantities ofamplified DNA are produced by the first reaction, a second PCR can beperformed to make multiple copies of DNA sequences of the firstamplified DNA. A nested set of primers are used in the second PCRreaction. The nested set of primers hybridize to sequences downstream ofthe 5′ primer and upstream of the 3′ primer used in the first reaction.

[0167] The present invention includes oligonucleotide which are usefulas primers for performing PCR methods to amplify the CRCA-1 transcriptor cDNA generated therefrom. According to the invention, diagnostic kitscan be assembled which are useful to practice methods of detecting thepresence of the CRCA-1 transcript or cDNA generated therefrom innon-colorectal samples. Such diagnostic kits comprise oligonucleotidewhich are useful as primers for performing PCR methods. It is preferredthat diagnostic kits according to the present invention comprise acontainer comprising a size marker to be run as a standard on a gel usedto detect the presence of amplified DNA. The size marker is the samesize as the DNA generated by the primers in the presence of the theCRCA-1 transcript or cDNA generated therefrom. Additional components insome kits include instructions for carrying out the assay. Additionallythe kit may optionally comprise depictions or photographs that representthe appearence of positive and negative results.

[0168] PCR assays are useful for detecting the CRCA-1 transcript inhomogenized tissue samples and cells in body fluid samples. It iscontemplated that PCR on the plasma portion of a fluid sample could beused to detect the CRCA-1 transcript.

[0169] Another method of determining whether a sample contains cellsexpressing CRCA-1 is by branched chain oligonucleotide hybridizationanalysis of mRNA extracted from a sample. Branched chain oligonucleotidehybridization may be performed as described in U.S. Pat. No. 5,597,909,U.S. Pat. No. 5,437,977 and U.S. Pat. No. 5,430,138, which are eachincorporated herein by reference. Reagents may be designed following theteachings of those patents and that sequence of the CCRA-1 transcript.

[0170] Another method of determining whether a sample contains cellsexpressing CRCA-1 is by Northern Blot analysis of mRNA extracted from anon-colorectal sample. The techniques for performing Northern blotanalyses are well known by those having ordinary skill in the art andare described in Sambrook, J. et al., (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. mRNA extraction, electrophoretic separation of the mRNA,blotting, probe preparation and hybridization are all well knowntechniques that can be routinely performed using readily availablestarting material.

[0171] The mRNA is extracted using poly dT columns and the material isseparated by electrophoresis and, for example, transferred tonitrocellulose paper. Labelled probes made from an isolated specificfragment or fragments can be used to visualize the presence of acomplementary fragment fixed to the paper. Probes useful to identifymRNA in a Northern Blot have a nucleotide sequence that is complementaryto the CRCA-1 transcript. Those having ordinary skill in the art coulduse the sequence information in SEQ ID NO:1 to design such probes or toisolate and clone the the CRCA-1 transcript or cDNA generated therefromto be used as a probe. Such probes are at least 15 nucleotides,preferably 30-200, more preferably 40-100 nucleotide fragments and maybe the entire CRCA-1 transcript.

[0172] According to the invention, diagnostic kits can be assembledwhich are useful to practice methods of detecting the presence of theCRCA-1 transcript in non-colorectal samples by Northern blot analysis.Such diagnostic kits comprise oligonucleotide which are useful as probesfor hybridizing to the mRNA. The probes may be radiolabelled. It ispreferred that diagnostic kits according to the present inventioncomprise a container comprising a size marker to be run as a standard ona gel. It is preferred that diagnostic kits according to the presentinvention comprise a container comprising a positive control which willhybridize to the probe. Additional components in some kits includeinstructions for carrying out the assay. Additionally the kit mayoptionally comprise depictions or photographs that represent theappearence of positive and negative results.

[0173] Northern blot analysis is useful for detecting the CRCA-1transcript in homogenized tissue samples and cells in body fluidsamples. It is contemplated that PCR on the plasma portion of a fluidsample could be used to detect the CRCA-1 transcript.

[0174] Another method of detecting the presence of the CRCA-1 transcriptby oligonucleotide hybridization technology. Oligonucleotidehybridization technology is well known to those having ordinary skill inthe art. Briefly, detectable probes which contain a specific nucleotidesequence that will hybridize to nucleotide sequence of the CRCA-1transcript. RNA or cDNA made from RNA from a sample is fixed, usually tofilter paper or the like. The probes are added and maintained underconditions that permit hybridization only if the probes fully complementthe fixed genetic material. The conditions are sufficiently stringent towash off probes in which only a portion of the probe hybridizes to thefixed material. Detection of the probe on the washed filter indicatecomplementary sequences.

[0175] Probes useful in oligonucleotide assays at least 18 nucleotidesof complementary DNA and may be as large as a complete complementarysequence to the CRCA-1 transcript. In some preferred embodiments theprobes of the invention are 30-200 nucleotides, preferably 40-100nucleotides. The probes preferably contain a sequence that is uniquewith respect to the sequence that encodes the ST receptor.

[0176] One having ordinary skill in the art, using the sequenceinformation disclosed in SEQ ID NO:1 can design probes which are fullycomplementary to the CRCA-1 transcript but not the sequence that encodesST receptor. Hybridization conditions can be routinely optimized tominimize background signal by non-fully complementary hybridization. Insome preferred embodiments, the probes are full length clones. Probesare at least 15 nucleotides, preferably 30-200, more preferably 40-100nucleotide fragments and may be the entire CRCA-1 transcript.

[0177] The present invention includes labelled oligonucleotide which areuseful as probes for performing oligonucleotide hybridization. That is,they are fully complementary with the CRCA-1 transcript but not the STreceptor transcript. For example, the mRNA sequence includes portionsencoded by different exons. The labelled probes of the present inventionare labelled with radiolabelled nucleotides or are otherwise detectableby readily available nonradioactive detection systems.

[0178] According to the invention, diagnostic kits can be assembledwhich are useful to practice oligonucleotide hybridization methods ofthe invention. Such diagnostic kits comprise a labelled oligonucleotidewhich encodes portions of the CRCA-1 transcript different from codingsequences that encode ST receptor. It is preferred that labelled probesof the oligonucleotide diagnostic kits according to the presentinvention are labelled with a radionucleotide. The oligonucleotidehybridization-based diagnostic kits according to the inventionpreferably comprise DNA samples that represent positive and negativecontrols. A positive control DNA sample is one that comprises a nucleicacid molecule which has a nucleotide sequence that is fullycomplementary to the probes of the kit such that the probes willhybridize to the molecule under assay conditions. A negative control DNAsample is one that comprises at least one nucleic acid molecule, thenucleotide sequence of which is partially complementary to the sequencesof the probe of the kit. Under assay conditions, the probe will nothybridize to the negative control DNA sample. Additional components insome kits include instructions for carrying out the assay. Additionallythe kit may optionally comprise depictions- or photographs thatrepresent the appearence of positive and negative results.

[0179] Oligonucleotide hybridization techniques are useful for detectingthe CRCA-1 transcript in homogenized tissue samples and cells in bodyfluid samples. It is contemplated that PCR on the plasma portion of afluid sample could be used to detect the CRCA-1 transcript.

[0180] The present invention relates to in vitro kits for evaluatingtissues samples to determine the level of metastasis and to reagents andcompositions useful to practice the same.

[0181] In some embodiments of the invention, tissue samples that includeportions of the lamina propria may be isolated from individualsundergoing or recovery from surgery to remove colorectal tumors includeresection or colonoscopy. The tissue is analyzed to identify thepresence or absence of the CRCA-1 transcript. Techniques such asimmunohistochemistry assays may be performed to determine whether theone or more CRCA-1 translation products is present in cells in thetissue sample which are indicative of metastatic migration. In someembodiments of the invention, tissue samples are analyzed to identifywhether the CRCA-1 is being expressed in cells in the tissue samplewhich indicate metastatic migration by detecting the presence or absenceof the CRCA-1 transcript or one or more CRCA-1 translation products. Thepresence of the CRCA-1 transcript or cDNA generated therefrom can bedetermined using techniques such as in situ hybridization orimmunohistochemistry.

[0182] The present invention relates to in vitro kits for evaluatingsamples of tumors to determine whether or not they are colorectal inorigin and to reagents and compositions useful to practice the same. Insome embodiments of the invention, tumor samples may be isolated fromindividuals undergoing or recovery from surgery to remove tumors in thecolon, tumors in other organs or biopsy material. The tumor sample isanalyzed to identify the presence or absence of the CRCA-1 transcript.Techniques such as immunohistochemistry assays may be performed todetermine whether one or more CRCA-1 translation products are present incells in the tumor sample which are indicative of colorectal origin.Alternatively, in some embodiments of the invention, lumen tissuesamples are analyzed to identify whether CRCA-1 is being expressed incells in the tumor sample which indicate colorectal origin by detectingthe presence or absence of the CRCA-1 transcript or one or more CRCA-1translation products. The presence of mRNA that encodes the ST receptorprotein or cDNA generated therefrom can be determined using techniquessuch as in situ hybridization, immunohistochemistry and in situ STbinding assay.

[0183] In situ hybridization technology is well known by those havingordinary skill in the art. Briefly, cells are fixed and detectableprobes which contain a specific nucleotide sequence are added to thefixed cells. If the cells contain complementary nucleotide sequences,the probes, which can be detected, will hybridize to them.

[0184] Probes useful in oligonucleotide assays at least 18 nucleotidesof complementary DNA and may be as large as a complete complementarysequence to the CRCA-1 transript. In some preferred embodiments theprobes of the invention are 30-200 nucleotides, preferably 40-100nucleotides. The probes contain a sequence that is unique from thosethat encode the ST receptor.

[0185] One having ordinary skill in the art, using the sequenceinformation set forth in SEQ ID NO:1 and the known sequence for human STreceptor mRNA can design probes useful in in situ hybridizationtechnology to identify cells that express CRCA-1. Probes preferablyhybridizes to a nucleotide sequence that corresponds to the CRCA-1transcript. Hybridization conditions can be routinely optimized tominimize background signal by non-fully complementary hybridization andcross hybridization to sequences encoding ST receptors. Probespreferably hybridize to the full length CRCA-1 transcript. Probes are atleast 15 nucleotides, preferably 30-200, more preferably 40-100nucleotide fragments and may be the CRCA transcript, more preferably18-28 nucleotide fragments of the The probes are fully complementary anddo not hybridize well to partially complementary sequences. For in situhybridization according to the invention, it is preferred that theprobes are detectable by fluorescence. A common procedure is to labelprobe with biotin-modified nucleotide and then detect with fluorescentlytagged avidin. Hence, probe does not itself have to be labelled withflorescent but can be subsequently detected with florescent marker.

[0186] The present invention includes labelled oligonucleotide which areuseful as probes for performing oligonucleotide hybridization. That is,they are fully complementary with mRNA sequences but not genomicsequences or ST recpetor mRNA. For example, the mRNA sequence includesportions encoded by different exons. The labelled probes of the presentinvention are labelled with radiolabelled nucleotides or are otherwisedetectable by readily available nonradioactive detection systems. Thepresent invention relates to probes useful for in situ hybridization toidentify cells that express CRCA-1.

[0187] Cells are fixed and the probes are added to the genetic material.Probes will hybridize to the complementary nucleic acid sequencespresent in the sample. Using a fluorescent microscope, the probes can bevisualized by their fluorescent markers.

[0188] According to the invention, diagnostic kits can be assembledwhich are useful to practice in situ hybridization methods of theinvention are fully complementary with mRNA sequences but not genomicsequences. For example, the mRNA sequence includes different exonsequences. It is preferred that labelled probes of the in situdiagnostic kits according to the present invention are labelled with afluorescent marker.

[0189] Those having ordinary skill in the art can analyze the fixedcells to characterize the level of metastatic migration of the coloncancer cells. The labelling of colon-derived cells allows for improvedanalysis.

[0190] Immunohistochemistry, techniques may be used to identify andessentially stain cells with one or more CRCA-1 translation products.Such “staining” allows for analysis of metastatic migration. Anti-CRCA-1translation product antibodies such as those described above ofcontacted with fixed cells and the CRCA-1 translation products presentin the cells reacts with the antibodies. The antibodies are detectablylabelled or detected using labelled second antibody or protein A tostain the cells.

[0191] The techniques described herein for evaluating tumor sections canalso be used to analyze tissue sections for samples of lymph nodes aswell as other tissues to identify the presence of colorectal tumorcells. The samples can be prepared and “stained” to detect expression ofCRCA-1.

[0192] In Vivo Imaging and Therapeutics

[0193] According to some embodiments of the invention, compositions andin vivo methods are provided for detecting, imaging, or treatingcolorectal tumors in an individual.

[0194] The conjugated compositions of the present invention are usefulfor targeting cells that line the inner intestine wall including cancercells derived from such cells, particularly metastasized cancer cellsderived from such cells.

[0195] When the conjugated compositions of the present invention areadministered outside the intestinal tract such as when administered inthe circulatory system, they remain segregated from the cells that linethe intestinal tract and will bind only to cells outside the intestinaltract which are derived from the intestinal tract such as metastasizedcolorectal cells. The conjugated compositions will not bind tonon-colorectal derived cells. Thus, the active moieties of conjugatedcompositions administered outside the intestinal tract are delivered tocells which are derived from the intestinal tract such as metastasizedcolorectal cells but will not be delivered to any other cells.

[0196] Therapeutic and diagnostic pharmaceutical compositions of thepresent invention include conjugated compounds specifically targeted tometastatic disease. These conjugated compounds include moieties thatbind to one or more CRCA-1 translation products which do not bind tocells of normal tissue in the body except cells of the intestinal tractsince the cells of other tissues do not possess such translationproducts. Further, according to the invention, the CRCA-1 translationproduct binding moieties do not bind to ST receptors.

[0197] Unlike normal colorectal cells and localized colorectal cancercells, metastasized colorectal cancer cells are accessible to substancesadministered outside the intestinal tract, for example administered inthe circulatory system. The only CRCA-1 translation products in normaltissue exist in the apical membranes of intestinal mucosa cells and thuseffectively isolated from the targeted cancer chemotherapeutics andimaging agents administered outside the intestinal tract by theintestinal mucosa barrier. Thus, metastasized colorectal cells may betargeted by conjugated compounds of the present invention by introducingsuch compounds outside the intestinal tract such as for example byadministering pharmaceutical compositions that comprise conjugatedcompounds into the circulatory system.

[0198] One having ordinary skill in the art can identify individualssuspected of suffering from colorectal cancer and metastasizedcolorectal cells. In those individuals diagnosed with colorectal cancer,it is standard therapy to suspect metastasis and aggressively attempt toeradicate metastasized cells. The present invention providespharmaceutical compositions and methods for imaging and thereby willmore definitively diagnose metastasis. Further, the present inventionprovides pharmaceutical compositions comprising therapeutic agents andmethods for specifically targeting and eliminating metastasizedcolorectal cancer cells. Further, the present invention providespharmaceutical compositions that comprise therapeutics and methods forspecifically eliminating colorectal cancer cells.

[0199] The pharmaceutical compositions which comprise conjugatedcompositions of the present invention may be used to diagnose or treatindividuals suffering from localized colorectal tumors, that is primaryor non-metastatic colorectal tumors if these have penetrated thebasement membrane underlying the mucosa into the submucosa where thereis abundant blood supply to which they have access. Penetration into thesubmucosa circumvents the mucosal barrier resulting in the ability ofconjugated compositions introduced into the circulatory system tointeract with these tumors.

[0200] The present invention relies upon the use of a CRCA-1 translationproduct binding moiety in a conjugated composition. The CRCA-1translation product binding moiety is essentially a portion of theconjugated composition which acts as a ligand to a CRCA-1 translationproduct and thus specifically binds to it. The conjugated compositionalso includes an active moiety which is associated with the CRCA-1translation product binding moiety; the active moiety being an activeagent which is either useful to image, target, neutralize or kill thecell.

[0201] According to the present invention, the CRCA-1 translationproduct binding moiety is the CRCA-1 translation product ligand portionof a conjugated composition. In some embodiments, the CRCA-1 translationproduct ligand is an antibody.

[0202] In some preferred embodiments, conjugated compounds compriseCRCA-1 translation product binding moieties that comprise an anti-CRCA-1translation product antibody.

[0203] It is preferred that the CRCA-1 translation product ligand usedas the CRCA-1 translation product binding moiety be as small aspossible. Thus it is preferred that the CRCA-1 translation productligand be a non-peptide small molecule or small peptide, preferably lessthan 25 amino acids, more preferably less than 20 amino acids. In someembodiments, the CRCA-1 translation product ligand which constitute theCRCA-1 translation product binding moiety of a conjugated composition isless than 15 amino acids. CRCA-1 translation product binding peptidecomprising less than 10 amino acids and CRCA-1 translation productbinding peptide less than 5 amino acids may be used as CRCA-1translation product binding moieties according to the present invention.It is within the scope of the present invention to include largermolecules which serve as CRCA-1 translation product binding moietiesincluding, but not limited to molecules such as antibodies whichspecifically bind to CRCA-1 translation product.

[0204] CRCA-1 translation product ligands useful as CRCA-1 translationproduct binding moieties may be identifed using various well knowncombinatorial library screening technologies such as those set forth inExample 1 herein.

[0205] An assay may be used to test both peptide and non-peptidecompositions to determine whether or not they are CRCA-1 translationproduct ligands or, to test conjugated compositions to determine if theypossess CRCA-1 translation product binding activity. Such compositionsthat specifically bind to CRCA-1 translation product can be identifiedby a competitive binding assay using antibodies known to bind to theCRCA-1 translation product. The competitive binding assay is a standardtechnique in pharmacology which can be readily performed by those havingordinary skill in the art using readily available starting materials.

[0206] CRCA-1 translation products may be produced synthetically,recombinantly or isolated from natural sources.

[0207] Using a solid phase synthesis as an example, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid supportare removed sequentially or concurrently, to provide the final peptide.The peptide of the invention are preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

[0208] CRCA-1 translation products and conjugated compositions orportions thereof which are peptides may also be prepared by recombinantDNA techniques. Provision of a suitable DNA sequence encoding thedesired peptide permits the production of the peptide using recombinanttechniques now known in the art. The coding sequence can be obtainedfrom natural sources or synthesized or otherwise constructed usingwidely available starting materials by routine methods. When the codingDNA is prepared synthetically, advantage can be taken of known codonpreferences of the intended host where the DNA is to be expressed.

[0209] To produce a CRCA-1 translation product which occurs in nature,one having ordinary skill in the art can, using well-known techniques,obtain a DNA molecule encoding the CRCA-1 translation product and insertthat DNA molecule into a commercially available expression vector foruse in well-known expression systems such as for example those describedherein.

[0210] For example, the commercially available plasmid pSE420(Invitrogen, San Diego, Calif.) may be used for recombinant productionin E. coli. The commercially available plasmid pYES2 (Invitrogen, SanDiego, Calif.) may be used for production in S. cerevisiae strains ofyeast. The commercially available MaxBac™ (Invitrogen, San Diego,Calif.) complete baculovirus expression system may be used forproduction in insect cells. The commercially available plasmid pcDNA I(Invitrogen, San Diego, Calif.) may be used for production in mammaliancells such as Chinese Hamster Ovary cells.

[0211] One having ordinary skill in the art may use these or othercommercially available expression vectors and systems or produce vectorsusing well-known methods and readily available starting materials.Expression systems containing the requisite control sequences, such aspromoters and polyadenylation signals, and preferably enhancers, arereadily available and known in the art for a variety of hosts. See e.g.,Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. ColdSpring Harbor Press (1989). Thus, the desired proteins can be preparedin both prokaryotic and eukaryotic systems, resulting in a spectrum ofprocessed forms of the protein.

[0212] The most commonly used prokaryotic system remains E. coli,although other systems such as B. subtilis and Pseudomonas are alsouseful. Suitable control sequences for prokaryotic systems include bothconstitutive and inducible promoters including the lac promoter, the trppromoter, hybrid promoters such as tac promoter, the lambda phage P1promoter. In general, foreign proteins may be produced in these hostseither as fusion or mature proteins. When the desired sequences areproduced as mature proteins, the sequence produced may be preceded by amethionine which is not necessarily efficiently removed. Accordingly,the peptides and proteins claimed herein may be preceded by anN-terminal Met when produced in bacteria. Moreover, constructs may bemade wherein the coding sequence for the peptide is preceded by anoperable signal peptide which results in the secretion of the protein.When produced in prokaryotic hosts in this matter, the signal sequenceis removed upon secretion.

[0213] A wide variety of eukaryotic hosts are also now available forproduction of recombinant foreign proteins. As in bacteria, eukaryotichosts may be transformed with expression systems which produce thedesired protein directly, but more commonly signal sequences areprovided to effect the secretion of the protein. Eukaryotic systems havethe additional advantage that they are able to process introns which mayoccur in the genomic sequences encoding proteins of higher organisms.Eukaryotic systems also provide a variety of processing mechanisms whichresult in, for example, glycosylation, carboxy-terminal amidation,oxidation or derivatization of certain amino acid residues,conformational control, and so forth.

[0214] Commonly used eukaryotic systems include, but are not limited to,yeast, fungal cells, insect cells, mammalian cells, avian cells, andcells of higher plants. Suitable promoters are available which arecompatible and operable for use in each of these host types as well asare termination sequences and enhancers, as e.g. the baculoviruspolyhedron promoter. As above, promoters can be either constitutive orinducible. For example, in mammalian systems, the mouse metallothionenepromoter can be induced by the addition of heavy metal ions.

[0215] The particulars for the construction of expression systemssuitable for desired hosts are known to those in the art. Forrecombinant production of the protein, the DNA encoding it is suitablyligated into the expression vector of choice and then used to transformthe compatible host which is then cultured and maintained underconditions wherein expression of the foreign gene takes place. Theprotein of the present invention thus produced is recovered from theculture, either by lysing the cells or from the culture medium asappropriate and known to those in the art.

[0216] One having ordinary skill in the art can, using well-knowntechniques, isolate the protein that is produced.

[0217] According to the present invention, the active moiety may be atherapeutic agent or an imaging agent. One having ordinary skill in theart can readily recognize the advantages of being able to specificallytarget metastasized colorectal cells with an CRCA-1 translation productligand and conjugate such a ligand with many different active agents.

[0218] Chemotherapeutics useful as active moieties which when conjugatedto a CRCA-1 translation product binding moiety are specificallydelivered to metastasized-colorectal cells are typically, small chemicalentities produced by chemical synthesis. Chemotherapeutics includecytotoxic and cytostatic drugs. Chemotherapeutics may include thosewhich have other effects on cells such as reversal of the transformedstate to a differentiated state or those which inhibit cell replication.Examples of chemotherapeutics include common cytotoxic or cytostaticdrugs such as for example: methotrexate (amethopterin), doxorubicin(adrimycin), daunorubicin, cytosinarabinoside, etoposide, 5-4fluorouracil, melphalan, chlorambucil, and other nitrogen mustards (e.g.cyclophosphamide), cis-platinum, vindesine (and other vinca alkaloids),mitomycin and bleomycin. Other chemotherapeutics include: purothionin(barley flour oligopeptide), macromomycin. 1,4-benzoquinone derivativesand trenimon.

[0219] Toxins are useful as active moieties. When a toxin is conjugatedto a CRCA-1 translation product binding moiety, the conjugatedcomposition is specifically delivered to a metastasized colorectal cellby way of the CRCA-1 translation product binding moiety and the toxinmoiety kills the cell. Toxins are generally complex toxic products ofvarious organisms including bacteria, plants, etc. Examples of toxinsinclude but are not limited to: ricin, ricin A chain (ricin toxin),Pseudomonas exotoxin (PE), diphtheria toxin (DT), Clostridiumperfringens phospholipase C (PLC), bovine pancreatic ribonuclease (BPR),pokeweed antiviral protein (PAP), abrin, abrin A chain (abrin toxin),cobra venom factor (CVF)., gelonin (GEL), saporin (SAP), modeccin,viscumin and volkensin. As discussed above, when protein toxins areemployed with CRCA-1 translation product binding peptides, conjugatedcompositions may be produced using recombinant DNA techniques. Briefly,a recombinant DNA molecule can be constructed which encodes both theCRCA-1 translation product ligand and the toxin on a chimeric gene. Whenthe chimeric gene is expressed, a fusion protein is produced whichincludes a CRCA-1 translation product binding moiety and an activemoiety. Protein toxins are also useful to form conjugated compounds withCRCA-1 translation product binding peptides through non-peptidyl bonds.

[0220] In addition, there are other approaches for utilizing activeagents for the treatment of cancer. For example, conjugated compositionsmay be produced which include a CRCA-1 translation product bindingmoiety and an active moiety which is an active enzyme. The CRCA-1translation product binding moiety specifically localizes the conjugatedcomposition to the tumor cells. An inactive prodrug which can beconverted by the enzyme into an active drug is administered to thepatient. The prodrug is only converted to an active drug by the enzymewhich is localized to the tumor. An example of an enzyme/prodrug pairincludes alkaline phosphatase/etoposidephosphate. In such a case, thealkaline phosphatase is conjugated to a CRCA-1 translation productbinding ligand. The conjugated compound is administered and localizes atthe metastasized cell. Upon contact with etoposidephosphate (theprodrug), the etoposidephosphate is converted to etoposide, achemotherapeutic drug which is taken up by the cancer cell.

[0221] Radiosensitizing agents are substances that increase thesensitivity of cells to radiation. Examples of radiosensitizing agentsinclude nitroimidazoles, metronidazole and misonidazole (see: DeVita, V.T. Jr. in Harrison's Principles of Internal Medicine, p.68, McGraw-HillBook Co., N.Y. 1983, which is incorporated herein by reference). Theconjugated compound that comprises a radiosensitizing agent as theactive moiety is administered and localizes at the metastasized cell.Upon exposure of the individual to radiation, the radiosensitizing agentis “excited” and causes the death of the cell.

[0222] Radionuclides may be used in pharmaceutical compositions that areuseful for radiotherapy or imaging procedures.

[0223] Examples of radionuclides useful as toxins in radiation therapyinclude: ⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au,²¹¹At, ²¹²Pb and ²¹²B. Other radionuclides which have been used by thosehaving ordinary skill in the art include: ³²P and ³³P, ⁷¹Ge, ⁷³As,¹⁰³Pb, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹⁰Sb, ¹²¹Sn, ¹³¹Cs ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os,^(193M)Pt, ¹⁹⁷Hg, all beta negative and/or auger emitters. Somepreferred radionuclides include: ⁹⁰Y, ¹³¹I ²¹¹At and ²¹²Pb/²¹²Bi.

[0224] According to the present invention, the active moieties may be animaging agent. Imaging agents are useful diagnostic procedures as wellas the procedures used to identify the location of metastasized cells.Imaging can be performed by many procedures well-known to those havingordinary skill in the art and the appropriate imaging agent useful insuch procedures may be conjugated to a CRCA-1 translation product ligandby well-known means. Imaging can be performed, for example, byradioscintigraphy, nuclear magnetic resonance imaging (MRI) or computedtomography (CT scan). The most commonly employed radionuclide imagingagents include radioactive iodine and indium. Imaging by CT scan mayemploy a heavy metal such as iron chelates. MRI scanning may employchelates of gadolinium or manganese. Additionally, positron emissiontomography (PET) may be possible using positron emitters of oxygen,nitrogen, iron, carbon, or gallium. Example of radionuclides useful inimaging procedures include: ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga ⁷⁷Br,⁸¹Rb/^(81M)Kr, ^(87M)Sr, ^(99M)Tc, ¹¹¹In, ^(113M)In, ¹²³I, ¹²⁵I, ¹²⁷Cs,¹²⁹Cs, ¹³¹I, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶Bi.

[0225] It is preferred that the conjugated compositions benon-immunogenic or immunogenic at a very low level. Accordingly, it ispreferred that the CRCA-1 translation product binding moiety be a small,poorly immunogenic or non-immunogenic peptide or a non-peptide.Alternatively, the CRCA-1 translation product binding moiety may be ahumanized or primatized antibody or a human antibody.

[0226] CRCA-1 translation product ligands are conjugated to activeagents by a variety of well-known techniques readily performed withoutundue experimentation by those having ordinary skill in the art. Thetechnique used to conjugate the CRCA-1 translation product ligand to theactive agent is dependent upon the molecular nature of the CRCA-1translation product ligand and the active agent. After the CRCA-1translation product ligand and the active agent are conjugated to form asingle molecule, assays may be performed to ensure that the conjugatedmolecule retains the activities of the moieties. The competitive bindingassay described above may be used to confirm that the CRCA-1 translationproduct binding moiety retains its binding activity as a conjugatedcompound. Similarly, the activity of the active moiety may be testedusing various assays for each respective type of active agent.Radionuclides retain there activity, i.e. their radioactivity,irrespective of conjugation. With respect to active agents which aretoxins, drugs and targeting agents, standard assays to demonstrate theactivity of unconjugated forms of these compounds may be used to confirmthat the activity has been retained.

[0227] Conjugation may be accomplished directly between the CRCA-1translation product ligand and the active agent or linking, intermediatemolecular groups may be provided between the CRCA-1 translation productligand and the active agent. Crosslinkers are particularly useful tofacilitate conjugation by providing attachment sites for each moiety.Crosslinkers may include additional molecular groups which serve asspacers to separate the moieties from each other to prevent either frominterfering with the activity of the other.

[0228] One having ordinary skill in the art may conjugate a CRCA-1translation product ligand to a chemotherapeutic drug using well-knowntechniques. For example, Magerstadt, M. Antibody Conjugates andMalignant Disease. (1991) CRC Press, Boca Raton, USA, pp. 110-152) whichis incorporated herein by reference, teaches the conjugation of variouscytostatic drugs to amino acids of antibodies. Such reactions may beapplied to conjugate chemotherapeutic drugs to CRCA-1 translationproduct ligands, including anti-CRCA-1 translation product antibodies,with an appropriate linker. Most of the chemotherapeutic agentscurrently in use in treating cancer possess functional groups that areamenable to chemical crosslinking directly with proteins. For example,free amino groups are available on methotrexate, doxorubicin,daunorubicin, cytosinarabinoside, cis-platin, vindesine, mitomycin andbleomycin while free carboxylic acid groups are available onmethotrexate, melphalan, and chlorambucil. These functional groups, thatis free amino and carboxylic acids, are targets for a variety ofhomobifunctional and heterobifunctional chemical crosslinking agentswhich can crosslink these drugs directly to the single free amino groupof an antibody. For example, one procedure for crosslinking CRCA-1translation product ligands which have a free amino group to activeagents which have a free amino group such as methotrexate, doxorubicin,daunorubicin, cytosinarabinoside, cis-platin, vindesine, mitomycin andbleomycin, or alkaline phosphatase, or protein- or peptide-based toxinemploys homobifunctional succinimidyl esters, preferably with carbonchain spacers such as disuccinimidyl suberate (Pierce Co, Rockford,Ill.). In the event that a cleavable conjugated compound is required,the same protocol would be employed utilizing 3,3′-dithiobis(sulfosuccinimidylpropionate; Pierce Co.).

[0229] In order to conjugate a CRCA-1 translation product ligand that isa peptiode or protein to a peptide-based active agent such as a toxin,the CRCA-1 translation product ligand and the toxin may be produced as asingle, fusion protein either by standard peptide synthesis orrecombinant DNA technology, both of which can be routinely performed bythose having ordinary skill in the art. Alternatively, two peptides, theCRCA-1 translation product ligand peptide and the peptide-based toxinmay be produced and/or isolated as separate peptides and conjugatedusing crosslinkers. As with conjugated compositions that containchemotherapeutic drugs, conjugation of CRCA-1 translation productbinding peptides and toxins can exploit the ability to modify the singlefree amino group of a CRCA-1 translation product binding peptide whilepreserving the receptor-binding function of this molecule.

[0230] One having ordinary skill in the art may conjugate a CRCA-1translation product ligand to a radionuclide using well-knowntechniques. For example, Magerstadt, M. (1991) Antibody Conjugates AndMalignant Disease, CRC Press, Boca Raton, Fla.,; and Barchel, S. W. andRhodes, B. H., (1983) Radioimaging and Radiotherapy, Elsevier, NY, N.Y.,each of which is incorporated herein by reference, teach the conjugationof various therapeutic and diagnostic radionuclides to amino acids ofantibodies.

[0231] The present invention provides pharmaceutical compositions thatcomprise the conjugated compounds of the invention and pharmaceuticallyacceptable carriers or diluents. The pharmaceutical composition of thepresent invention may be formulated by one having ordinary skill in theart. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, A. Osol, a standard reference text in thisfield, which is incorporated herein by reference. In carrying outmethods of the present invention, conjugated compounds of the presentinvention can be used alone or in combination with other diagnostic,therapeutic or additional agents. Such additional agents includeexcipients such as coloring, stabilizing agents, osmotic agents andantibacterial agents. Pharmaceutical compositions are preferably sterileand pyrogen free.

[0232] The conjugated compositions of the invention can be, for example,formulated as a solution, suspension or emulsion in association with apharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Liposomes may also be used. The vehicle may containadditives that maintain isotonicity (e.g., sodium chloride, mannitol)and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques. For example, aparenteral composition suitable for administration by injection isprepared by dissolving 1.5% by weight of active ingredient in 0.9%sodium chloride solution.

[0233] The pharmaceutical compositions according to the presentinvention may be administered as either a single dose or in multipledoses. The pharmaceutical compositions of the present invention may beadministered either as individual therapeutic agents or in combinationwith other therapeutic agents. The treatments of the present inventionmay be combined with conventional therapies, which may be administeredsequentially or simultaneously.

[0234] The pharmaceutical compositions of the present invention may beadministered by any means that enables the conjugated composition toreach the targeted cells. In some embodiments routes of administrationinclude those selected from the group consisting of intravenous,intraarterial, intraperitoneal, local administration into the bloodsupply of the organ in which the tumor resides or directly into thetumor itself. Intravenous administration is the preferred mode ofadministration. It may be accomplished with the aid of an infusion pump.

[0235] The dosage administered varies depending upon factors such as:the nature of the active moiety; the nature of the conjugatedcomposition; pharmacodynamic characteristics; its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms; kind of concurrent treatment; and frequency oftreatment.

[0236] Because conjugated compounds are specifically targeted to cellswith one or more CRCA-1 translation products, conjugated compounds whichcomprise chemotherapeutics or toxins are administered in doses less thanthose which are used when the chemotherapeutics or toxins areadministered as unconjugated active agents, preferably in doses thatcontain up to 100 times less active agent. In some embodiments,conjugated compounds which comprise chemotherapeutics or toxins areadministered in doses that contain 10-100 times less active agent as anactive moiety than the dosage of chemotherapeutics or toxinsadministered as unconjugated active agents. To determine the appropriatedose, the amount of compound is preferably measured in moles instead ofby weight. In that way, the variable weight of different CRCA-1translation product binding moieties does not affect the calculation.Presuming a one to one ratio of CRCA-1 translation product bindingmoiety to active moiety in conjugated compositions of the invention,less moles of conjugated compounds may be administered as compared tothe moles of unconjugated compounds administered, preferably up to 100times less moles.

[0237] Typically, chemotherapeutic conjugates are administeredintravenously in multiple divided doses.

[0238] Up to 20 gm IV/dose of methotrexate is typically administered inan unconjugated form. When methotrexate is administered as the activemoiety in a conjugated compound of the invention, there is a 10-to100-fold dose reduction. Thus, presuming each conjugated compoundincludes one molecule of methotrexate conjugated to one CRCA-1translation product binding moiety, of the total amount of conjugatedcompound administered, up to about 0.2-2.0 g of methotrexate is presentand therefore administered. In some embodiments, of the total amount ofconjugated compound administered, up to about 200 mg-2 g of methotrexateis present and therefore administered.

[0239] To dose conjugated compositions comprising CRCA-1 translationproduct binding moieties linked to active moieties that areradioisotopes in pharmaceutical compositions useful as imaging agents,it is presumed that each CRCA-1 translation product binding moiety islinked to one radioactive active moiety. The amount of radioisotope tobe administered is dependent upon the radioisotope. Those havingordinary skill in the art can readily formulate the amount of conjugatedcompound to be administered based upon the specific activity and energyof a given radionuclide used as an active moiety. Typically 0.1-100millicuries per dose of imaging agent, preferably 1-10 millicuries, mostoften 2-5 millicuries are administered. Thus, pharmaceuticalcompositions according to the present invention useful as imaging agentswhich comprise conjugated compositions comprising a CRCA-1 translationproduct binding moiety and a radioactive moiety comprise 0.1-100millicuries, in some embodiments preferably 1-10 millicuries, in someembodiments preferably 2-5 millicuries, in some embodiments morepreferably 1-5 millicuries. Examples of dosages include: ¹³¹I=betweenabout 0.1-100 millicuries per dose, in some embodiments preferably 1-10millicuries, in some embodiments 2-5 millicuries, and in someembodiments about 4 millicuries; ¹¹¹In =between about 0.1-100millicuries per dose, in some embodiments preferably 1-10 millicuries,in some embodiments 1-S millicuries, and in some embodiments about 2millicuries; ^(99m)Tc=between about 0.1-100 millicuries per dose, insome embodiments preferably 5-75 millicuries, in some embodiments 10-50millicuries, and in some embodiments about 27 millicuries. Wessels B. W.and R. D. Rogus (1984) Med. Phys. 11:638 and Kwok, C. S. et al. (1985)Med. Phys. 12:405, both of which are incorporated herein by reference,disclose detailed dose calculations for diagnostic and therapeuticconjugates which may be used in the preparation of pharmaceuticalcompositions of the present invention which include radioactiveconjugated compounds.

[0240] One aspect of the present invention relates to a method oftreating individuals suspected of suffering from metastasized colorectalcancer. Such individuals may be treated by administering to theindividual a pharmaceutical composition that comprises apharmaceutically acceptable carrier or diluent and a conjugated compoundthat comprises a CRCA-1 translation product binding moiety and an activemoiety wherein the active moiety is a radiostable therapeutic agent. Insome embodiments of the present invention, the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier or diluentand a conjugated compound that comprises a CRCA-1 translation productbinding moiety and an active moiety wherein the active moiety is aradiostable active agent and the CRCA-1 translation product bindingmoiety is an antibody. In some embodiments of the present invention, thepharmaceutical composition comprises a pharmaceutically acceptablecarrier or diluent and a conjugated compound that comprises a CRCA-1translation product binding moiety and an active moiety wherein theactive moiety is a radiostable therapeutic agent. In some embodiments ofthe present invention, the pharmaceutical composition comprises apharmaceutically acceptable carrier or diluent and a conjugated compoundthat comprises a CRCA-1 translation product binding moiety and an activemoiety wherein the active moiety is a radiostable active agent selectedfrom the group consisting of: methotrexate, doxorubicin, daunorubicin,cytosinarabinoside, etoposide, 5-4 fluorouracil, melphalan,chlorambucil, cis-platinum, vindesine, mitomycin, bleomycin,purothionin, macromomycin, 1,4-benzoquinone derivatives, trenimon,ricin, ricin A chain, Pseudomnas exotoxin, diphtheria toxin, Clostridiumperfringens phospholipase C, bovine pancreatic ribonuclease, pokeweedantiviral protein, abrin, abrin A chain, cobra venom factor, gelonin,saporin, modeccin, viscumin, volkensin, alkaline phosphatase,nitroimidazole, metronidazole and misonidazole. The individual beingtreated may be diagnosed as having metastasized colorectal cancer or maybe diagnosed as having localized colorectal cancer and may undergo thetreatment proactively in the event that there is some metastasis as yetundetected. The pharmaceutical composition contains a therapeuticallyeffective amount of the conjugated composition. A therapeuticallyeffective amount is an amount which is effective to cause a cytotoxic orcytostatic effect on metastasized colorectal cancer cells withoutcausing lethal side effects on the individual.

[0241] One aspect of the present invention relates to a method oftreating individuals suspected of suffering from metastasized colorectalcancer. Such individuals may be treated by administering to theindividual a pharmaceutical composition that comprises apharmaceutically acceptable carrier or diluent and a conjugated compoundthat comprises a CRCA-1 translation product binding moiety and an activemoiety wherein the active moiety is a radioactive. In some embodimentsof the present invention, the pharmaceutical composition comprises apharmaceutically acceptable carrier or diluent and a conjugated compoundthat comprises a CRCA-1 translation product binding moiety and an activemoiety wherein the active moiety is a radioactive and the ST receptorbinding moiety is an antibody. In some embodiments of the presentinvention, the pharmaceutical composition comprises a pharmaceuticallyacceptable carrier or diluent and a conjugated compound that comprises aCRCA-1 translation product and an active moiety wherein the activemoiety is a radioactive agent selected from the group consisting of:⁴⁷SC, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At,²¹²Pb, ²¹²B, ³²P and ³³P, ⁷¹Ge, ⁷⁷As, ¹⁰³Pb, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn,¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os ^(193M)Pt, ¹⁹⁷Hg, ³²P and ³³P, ⁷¹Ge,⁷⁷As, ¹⁰³Pb, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu,¹⁹¹Os, ^(193M)Pt, ¹⁹⁷Hg, all beta negative and/or auger emitters. Theindividual being treated may be diagnosed as having metastasizedcolorectal cancer or may be diagnosed as having localized colorectalcancer and may undergo the treatment proactively in the event that thereis some metastasis as yet undetected. The pharmaceutical compositioncontains a therapeutically effective amount of the conjugatedcomposition. A therapeutically effective amount is an amount which iseffective to cause a cytotoxic or cytostatic effect on metastasizedcolorectal cancer cells without causing lethal side effects on theindividual.

[0242] One aspect of the present invention relates to a method ofdetecting metastasized colorectal cancer cells in an individualsuspected of suffering from metastasized colorectal cancer byradioimaging. Such individuals may be diagnosed as suffering frommetastasized colorectal cancer and the metastasized colorectal cancercells may be detected by administering to the individual, preferably byintravenous administration, a pharmaceutical composition that comprisesa pharmaceutically acceptable carrier or diluent and a conjugatedcompound that comprises a CRCA-1 translation product binding moiety andan active moiety wherein the active moiety is a radioactive anddetecting the presence of a localized accumulation or aggregation ofradioactivity, indicating the presence of cells with a CRCA-1translation product. In some embodiments of the present invention, thepharmaceutical composition comprises a pharmaceutically acceptablecarrier or diluent and a conjugated compound that comprises a CRCA-1translation product binding moiety and an active moiety wherein theactive moiety is a radioactive and the ST receptor binding moiety is anantibody. In some embodiments of the present invention, thepharmaceutical composition comprises a pharmaceutically acceptablecarrier or diluent and a conjugated compound that comprises an STreceptor binding moiety and an active moiety wherein the active moietyis a radioactive agent selected from the group consisting of:radioactive heavy metals such as iron chelates, radioactive chelates ofgadolinium or manganese, positron emitters of oxygen, nitrogen, iron,carbon, or gallium, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br,⁸¹Rb/^(81M)Kr, ⁸⁷Sr, ^(99M)Tc, ¹¹¹In, ^(113M)In, ¹²³I, ¹²⁵I, ¹²⁷Cs,¹²⁹Cs, ¹³¹I, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶Bi. The individual being treatedmay be diagnosed as having metastasized colorectal cancer or may bediagnosed as having localized colorectal cancer and may undergo thetreatment proactively in the event that there is some metastasis as yetundetected. The pharmaceutical composition contains a diagnosticallyeffective amount of the conjugated composition. A diagnosticallyeffective amount is an amount which can be detected at a site in thebody where cells with ST receptors are located without causing lethalside effects on the individual.

[0243] Another aspect of the invention relates to unconjugatedcompositions which comprise a CRCA-1 translation product binding ligandand an active agent. For example, liposomes are small vesicles composedof lipids. Drugs can be introduced into the center of these vesicles.The outer shell of these vesicles comprise a CRCA-1 translation productbinding ligand. Liposomes Volumes 1, 2 and 3 CRC Press Inc. Boca RatonFla., which is incorporated herein by reference, disclose preparation ofliposome-encapsulated active agents which include targeting agents thatcorrespond to CRCA-1 translation product ligand in the outer shell.Unconjugated compositions which comprise a CRCA-1 translation productligand in the matrix of a liposome with an active agent inside includesuch compositions in which the CRCA-1 translation product ligand is sanantibody and the active agent is selected from the group consisting of:methotrexate, doxorubicin, daunorubicin, cytosinarabinoside, etoposide,5-4 fluorouracil, melphalan, chlorambucil, cis-platinum, vindesine,mitomycin, bleomycin, purothionin, macromomycin, 1,4-benzoquinonederivatives, trenimon, ricin, ricin A chain, Pseudomonas exotoxin,diphtheria toxin, Clostridium perfringens phospholipase C, bovinepancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin Achain, cobra venom factor, gelonin, saporin, modeccin, viscumin,volkensin, alkaline phosphatase, nitroimidazole metronidazole andmisonidazole.

[0244] Drug Delivery Targeted to Colon Cells Generally

[0245] Another aspect of the invention relates to unconjugated andconjugated compositions which comprise a CRCA-1 translation productligand used to deliver therapeutic nucleic acid molecules to cells thatcomprise a CRCA-1 translation product such as normal and cancer cells ofthe intestinal tract as well as metastasized colorectal cancer cells. Insome embodiments, the genetic material is delivered to metastasizedtumor cells to produce an antigen that can be targeted by the immunesystem or to produce a protein which kills the cell or inhibits itsproliferation. In some embodiments, the CRCA-1 translation productligand is used to deliver nucleic acids that encode nucleic acidmolecules which replace defective endogenous genes or which encodetherapeutic proteins. In some embodiments, the CRCA-1 translationproduct ligand is thus used to deliver the active agent specifically tothe cells lining the intestinal tract to treat diseases specific to thisorgan. According to this aspect of the invention, compositions comprisenucleic acid molecules which can replace defective genes. In someembodiments, the compositions are used in gene therapy protocols todeliver to individuals, genetic material needed and/or desired to makeup for a genetic deficiency.

[0246] In some embodiments, the CRCA-1 translation product ligand iscombined with or incorporated into a delivery vehicle thereby convertingthe delivery vehicle into a specifically targeted delivery vehicle. Forexample, a CRCA-1 translation product binding peptide may be integratedinto the outer portion of a viral particle making such a virus a CRCA-1translation product-bearing cell specific virus. Similarly, the coatprotein of a virus may be engineered such that it is produced as afusion protein which includes an active CRCA-1 translation productbinding peptide that is exposed or otherwise accessible on the outsideof the viral particle making such a virus a CRCA-1 translationproduct-bearing cell-specific virus. In some embodiments, a CRCA-1translation product ligand may be integrated or otherwise incorporatedinto the liposomes wherein the CRCA-1 translation product ligand isexposed or otherwise accessible on the outside of the liposome makingsuch liposomes specifically targeted to CRCA-1 translationproduct-bearing cells.

[0247] The active agent in the conjugated or unconjugated compositionsaccording to this aspect of the invention is a nucleic acid molecule.The nucleic acid may be RNA or preferably DNA. In some embodiments, thenucleic acid molecule is an antisense molecule or encodes an antisensesequence whose presence in the cell inhibits production of anundesirable protein. In some embodiments, the nucleic acid moleculeencodes a ribozyme whose presence in the cell inhibits production of anundesirable protein. In some embodiments, the nucleic acid moleculeencodes a protein or peptide that is desirably produced in the cell. Insome embodiments, the nucleic acid molecule encodes a functional copy ofa gene that is defective in the targeted cell. The nucleic acid moleculeis preferably operably linked to regulatory elements needed to expressthe coding sequence in the cell.

[0248] Liposomes are small vesicles composed of lipids. Geneticconstructs which encode proteins that are desired to be expressed inCRCA-1 translation product-bearing cells are introduced into the centerof these vesicles. The outer shell of these vesicles comprise an aCRCA-1 translation product ligand. Liposomes Volumes 1, 2 and 3 CRCPress Inc. Boca Raton Fla., which is incorporated herein by reference,disclose preparation of liposome-encapsulated active agents whichinclude antibodies in the outer shell. In the present invention, aCRCA-1 translation product ligand such as for example an anti-CRCA-1translation product antibodies is associated with the in the outershell. Unconjugated compositions which comprise a CRCA-1 translationproduct ligand in the matrix of a liposome with an active agent insideinclude such compositions in which the CRCA-1 translation product ligandis preferably an antibody.

[0249] In one embodiment for example, cystic fibrosis, a genetic diseasein which there is a mutation of a specific gene encoding a chloridetransport protein which ultimately produces abnormalities of function inmany systems, most notably in the respiratory and intestinal tract, istreated by gene therapy techniques using CRCA-1 translation productligands to deliver the corrective gene to cells. Current therapy hasbeen directed at replacing the mutant gene in the respiratory systemwith the normal gene by targeting these genes directly to the cellslining the respiratory tract using viruses which bind only to thosecells. Similarly, the normal gene is packaged in liposomes targeted ontheir surface with CRCA-1 translation product ligands and delivered tothe intestinal tract. CRCA-1 translation product ligands specificallytarget and direct the liposomes containing the normal gene to correctthe lesion for cystic fibrosis to the specific cells lining theintestinal tract, from the duodenum to the rectum. Uptake of thatgenetic material by those cells should result in a cure of cysticfibrosis in the intestinal tract.

[0250] In another embodiment, the delivery of normal copies of the p53tumor suppressor gene to the intestinal tract is accomplished usingCRCA-1 translation product ligand to target the gene therapeutic.Mutations of the p53 tumor suppressor gene appears to play a prominentrole in the development of colorectal cancer in the intestinal tract.One approach to combatting this disease is the delivery of normal copiesof this gene to the intestinal tract to cells expressing mutant forms ofthis gene. Genetic constructs that comprise normal p53 tumor suppressorgenes are incorporated into liposomes that comprise a CRCA-1 translationproduct ligand. The composition is delivered to the intestinal tract.CRCA-1 translation product binding ligands specifically target anddirect the liposomes containing the normal gene to correct the lesioncreated by mutation of p53 suppressor gene in intestinal cells.

[0251] Preparation of genetic constructs is with the skill of thosehaving ordinary skill in the art. The present invention allows suchconstruct to be specifically targeted by using the CRCA-1 translationproduct ligands of the present invention. The compositions of theinvention include a CRCA-1 translation product ligand such as ananti-CRCA-1 translation product antibody associated with a deliveryvehicle and a gene construct which comprises a coding sequence for aprotein whose production is desired in the cells of the intestinal tractlinked to necessary regulatory sequences for expression in the cells.For uptake by cells of the intestinal tract, the compositions areadministered orally or by enema whereby they enter the intestinal tractand contact cells which comprise one or more CRCA-1 translationproducts. The delivery vehicles associate with the CRCA-1 translationproduct by virtue of the CRCA-1 translation product ligand and thevehicle is internalized into the cell or the active agent/geneticconstruct is otherwise taken up by the cell. Once internalized, theconstruct can provide a therapeutic effect on the individual. One havingordinary skill in the art can readily formulate such compositions fororal or enema administration and determine the effective amount of suchcomposition to be administered to treat the disease or disorder.

[0252] Antisense

[0253] The present invention provides compositions, kits and methodswhich are useful to prevent and treat diseases effecting colon cells byproviding the means to specifically deliver antisense compounds to coloncells and thereby stop expression of genes in such colon cells in whichundesirable gene expression is taking place without negatively effectingcells in which no such expression occurs.

[0254] The conjugated compositions of the present invention are usefulfor targeting cells that line the inner intestine wall including thosecancer cells derived from such cells, including metastasized cancercells as well as localized cancer and normal colon cells. The conjugatedcompositions will not bind to non-colorectal derived cells. Thus, theactive moieties of conjugated compositions administered to an individualare delivered to cells which are derived from the intestinal tract suchas local normal and cancerous colorectal cells and metastasizedcolorectal cells. Non-colorectal cells, lacking one or more CRCA-1translation products, do not take up the conjugated compositions. Thus,the present invention provides compositions and methods of deliveringantisense compositions to colon cells only.

[0255] The present invention provides a colorectal cancer specificapproach in which only colorectal cells are exposed to the activeportion of the compound and only colorectal cancer cells are effected bythe conjugated compound. The ST receptor binding moiety specificallybinds to colorectal cells, including normal colorectal cells, localizedcolorectal cancer cells and metastasized colorectal cancer cells. Uponbinding to these cells, the conjugated compound is internalized and thedelivery of the conjugated compound including the antisense portion ofthe molecule is effected. The presence of the conjugated compound innormal colorectal cells has no effect on such cells because thecolorectal cancer-associated gene for which the antisense molecule thatmakes up the active moiety of the conjugated compound is complementaryis not being expressed. However, in colorectal cancer cells, the cancergene for which the antisense molecule that makes up the active moiety ofthe conjugated compound is complementary is being expressed. Thepresence of the conjugated compound in colorectal cancer cells serves toinhibit or prevent transcription or translation of the cancer gene andthereby reduce or eliminate the transformed phenotype.

[0256] The invention can be used to combat localized or metastasizedcolorectal cancer as well as to prevent the emergence of the transformedphenotype. Thus the invention can be used therapeutically as well asprophylactically.

[0257] Therapeutic and prophylactic pharmaceutical compositions of thepresent invention include conjugated compounds specifically targeted tocolon cells. These conjugated compounds include CRCA-1 translationproducts binding moieties which do not bind to cells of normal tissue inthe body except cells of the intestinal tract since the cells of othertissues do not possess ST receptors. Thus, only normal colorectal cells,localized colorectal cancer cells and metastasized colorectal cancercells take up the conjugated compositions.

[0258] One having ordinary skill in the art can readily identifyindividuals suspected of suffering from colorectal cancer andmetastasized colorectal cells. In those individuals diagnosed withcolorectal cancer, it is standard therapy to suspect metastasis andaggressively attempt to eradicate metastasized cells. The presentinvention provides pharmaceutical compositions and methods forspecifically targeting and eliminating metastasized colorectal cancercells. Further, the present invention provides pharmaceuticalcompositions that comprise therapeutics and methods for specificallyeliminating colorectal cancer cells. The present invention providespharmaceutical compositions and methods for specifically in colorectalcells and preventing transformation by such cells by prophylacticallyfurnishing such cells with antisense molecules that inhibittranscription or translation of genes involved in transformation.

[0259] The pharmaceutical compositions which comprise conjugatedcompositions of the present invention may be used to diagnose or treatindividuals suffering from localized and/or metastatic colorectaltumors, that is primary or non-metastatic colorectal tumors as well asmetastasized colorectal tumors.

[0260] The present invention relies upon the use of a CRCA-1 translationproduct binding moiety in a conjugated composition. The CRCA-1translation product binding moiety is essentially a portion of theconjugated composition which acts as a ligand to the CRCA-1 translationproduct and thus specifically binds to these receptors. The conjugatedcomposition also includes an active moiety which is associated with theCRCA-1 translation product binding moiety; the active moiety being anantisense composition useful to inhibit or prevent transcription ortranslation of expression of genes whose expression is associated withcancer.

[0261] According to the present invention, the active moiety is anantisense composition. In particular, the antisense molecule that makesup the active moiety of a conjugated compound hybridizes to DNA or RNAin a colon cell and inhibits and/or prevents transcription ortranslation of the DNA or RNA from taking place. The antisensecompositions may be a nucleic acid molecule, a derivative or an analogsthereof. The chemical nature of the antisense composition may be that ofa nucleic acid molecule or a modified nucleic acid molecule or anon-nucleic acid molecule which possess functional groups that mimic aDNA or RNA molecule that is complementary to the DNA or RNA moleculewhose expression is to be inhibited or otherwise prevented. Antisensecompositions inhibit or prevent transcription or translation of geneswhose expression is linked to colorectal cancer, i.e. colorectal cancerassociated genes. Examples of such genes include, but are not limitedto: hereditary nonpolyposis coli (HNPCC) genes such as hMSH2, hMLH1,hPMS1, and hPMS2, Ras, adenomatous polyposis coli (APC), ERBB-1/HER-1,ERBB-2/HER-2, p53 Tumor Suppressor, MYB, FOS, ABL, MYC, Protein TyrosinePhosphatase G1, Cyclic AMP-Dependent Protein Kinase (PKA), CRIPTO,Transforming Growth Factor Alpha and 1p.

[0262] Colorectal cancer-associated genes provide the genetic basis forcolorectal cancer. Colorectal cancer is a process involving accumulationof genetic mutations in epithelial cells leading to the neoplasticphenotype associated with unregulated growth. Colorectal carcinogenesisis a multistage process involving the progression from adenomas toinvasive carcinomas. Indeed, the cumulative total of geneticabnormalities appear to be more important than their order ofappearance. Many of the genetic abnormalities result from allelic lossor deletion of fragments of chromosomes. Specific genetic abnormalitieswhich have been associated with the colorectal cancer phenotype will bediscussed below. These all are potential s for treatment employinggenetic approaches. See: Toribara, N W and Sleisenger, M H (1995)Screening for colorectal cancer. New Eng. J. Med. 332:861-867 which ishereby incorporated herein by reference including all references citedtherein which are also hereby incorporated herein by reference.

[0263] HNPCC refers to 4 genes that are suspected to be responsible forhereditary nonpolyposis coli (HNPCC) colorectal cancer. These four geneshave been identified and are discussed in Toribara, N W and Sleisenger,M H (1995) Screening for colorectal cancer. New Eng. J. Med.332:861-867. The genes, called hMSH2, hMLH1, hPMS1, and hPMS2, areproofreading genes that repair mismatches of bases in DNA. Loss of thisfunction allows replication errors to occur in the DNA.

[0264] Point mutations insertions, and deletions in K-ras and H-ras havebeen identified in colorectal tumors. See: Toribara, N W and Sleisenger,M H (1995) Screening for colorectal cancer. New Eng. J. Med.332:861-867; Kniazev, P G, et al. Complex characteristics of thealterations of oncogenes HER-2/ERBB-2, HER-l/ERBB-1, HRAS-1, C-MYC andanti-oncogenes p53, RB1, as well as deletions of loci of chromosome 17in colon carcinoma. Molekuliarnaia Biologiia. 26(5):1134-47, 1992,Sep-Oct. and Ramsay, R G, et al. Myb expression is higher in malignanthuman colonic carcinoma and premalignant adenomatous polyps than innormal mucosa. Cell Growth & Differentiation. 3(10):723-30, 1992 Oct.,.which are each hereby incorporated herein by reference including allreferences cited therein which are also hereby incorporated herein byreference.

[0265] Patients with familial adenomatous polyposis coli (APC) appear tohave a series of deletions including deletions of chromosome 5q, 18q,17p. The 17p deletion represents a deletion of the p53 suppressor gene.

[0266] ERBB-1/HER-1 and ERBB-2/HER-2 genes have been demonstrated to beamplified in about 4-8% of cases of colorectal cancer.

[0267] Point mutations in p53 genes have been reported to be mutated inabout 3% of colorectal cancer cases.

[0268] MYB proto-oncogene expression has been demonstrated to be higherin colorectal tumors. See: Ramsay, R G, Thompson, M A, Hayman, J A,Reid, G, Gonda, T J, Whitehead, R H. Myb expression is higher inmalignant human colonic carcinoma and premalignant adenomatous polypsthan in normal mucosa. Cell Growth & Differentiation. 3(10):723-30, 1992Oct.; Melani, C. et al. Inhibition of proliferation by c-myb antisenseoligodeoxynucleotides in colon adenocarcinoma cell lines that expressc-myb. Cancer Research 51(11):2897-901, 1991, Jun 1; and Ramsay R G, etal. Myb expression is higher in malignant human colonic carcinoma andpremalignant adenomatous polyps than in normal mucosa. Cell Growth &Differentiation. 3(10):723-30, 1992 Oct.; which are each herebyincorporated herein by reference including all references cited thereinwhich are also hereby incorporated herein by reference. Indeed, tumorsand cells with the highest levels of expression of MYB were the mostdysplastic and had the highest levels of proliferation. cMYB is aprotooncogene which plays a role in the proliferation signaling pathway.Rearrangements, insertions, and deletions of this gene have beenobserved. See: Alexander, R J, et al. Oncogene alterations in rat colontumors induced by N-methyl-N-nitrosourea. American Journal of theMedical Sciences. 303(1):16-24, 1992, Jan. which is hereby incorporatedherein by reference including all references cited therein which arealso hereby incorporated herein by reference. Antisense MYBoligonucleotides retard the proliferation of colonic adenocarcinomacells which had the highest level of expression of this oncogene, invitro.

[0269] Chemical carcinogenesis in a rat model demonstrated pointmutations in fos, an oncogene which mediates transcriptional regulationand proliferation. See: Alexander, R J, et al. Oncogene alterations inrat colon tumors induced by N-methyl-N-nitrosourea. American Journal ofthe Medical Sciences. 303(1):16-24, 1992, Jan. which is herebyincorporated herein by reference including all references cited thereinwhich are also hereby incorporated herein by reference.

[0270] Chemical carcinogenesis in a rat model demonstrated pointmutations in the oncogene abl. See: Alexander, R J, et al. Oncogenealterations in rat colon tumors induced by N-methyl-N-nitrosourea.American Journal of the Medical Sciences. 303(1):16-24, 1992, Jan.

[0271] MYC is an oncogene that plays a role in regulating transcriptionand proliferation. Increased expression of MYC has been found incolorectal cancer cells. Collins, J F, et al. c-myc antisenseoligonucleotides inhibit the colony-forming capacity of Colo 320 coloniccarcinoma cells. Journal of Clinical Investigation. 89(5):1523-7, 1992May.; and Rodriguez-Alfageme, C, et al. Suppression of deregulated c-MYCexpression in human colon carcinoma cells by chromosome 5 transfer.Proceedings of the National Academy of Sciences of the United States ofAmerica. 89(4):1482-6, 1992 Feb 15. which are both hereby incorporatedherein by reference including all references cited therein which arealso hereby incorporated herein by reference. A 15-base antisenseoligonucleotide to myc complementary to the translation initiationregion of exon II was incubated with colorectal cancer cells. Thisantisense molecule inhibited proliferation of colorectal cancer cells ina dos-dependent fashion. Interestingly, the uptake of thisoligonucleotide was low (0.7%). Also, transfer of a normal chromosome 5to colorectal cancer cells resulted in the regulation of myc expressionand loss of proliferation. These data suggest that a tumor suppressorgene important in the regulation of myc is contained on this chromosome.

[0272] A novel protein tyrosine phosphatase, G1, has been identified.Examination of the mRNA encoding this protein in colorectal tumor cellsrevealed that it undergoes point mutations and deletions in these cellsand may play a role in proliferation characteristic of these cells.Takekawa, M. et al. Chromosomal localization of the protein tyrosinephosphatase G1 gene and characterization of the aberrant transcripts inhuman colon cancer cells. FEBS Letters. 339(3):222-8, 1994 Feb. 21,which is hereby incorporated herein by reference including allreferences cited therein which are also hereby incorporated herein byreference.

[0273] Gastrin regulates colon cancer cell growth through a cyclicAMP-dependent mechanism mediated by PKA. Antisense oligodeoxynucleotidesto the regulatory subunit of a specific class of PKA inhibited thegrowth-promoting effects of cyclic AMP in colon carcinoma cells. See:Bold, R J, et al. Experimental gene therapy of human colon cancer.Surgery. 116(2)—189-95; discussion 195-6, 1994 Aug. and Yokozaki, H., etal. An antisense oligodeoxynucleotide that depletes RI alpha subunit ofcyclic AMP-dependent protein kinase induces growth inhibition in humancancer cells. Cancer Research. 53(4):868-72, 1993 Feb. 15, which areboth hereby incorporated herein by reference including all referencescited therein which are also hereby incorporated herein by reference.

[0274] CRIPTO is an epidermal growth factor-related gene expressed in amajority of colorectal cancer tumors. Antisense phosphorothioateoligodeoxynucleotides to the 5′-end of CRIPTO mRNA significantly reducedCRIPTO expression and inhibited colorectal tumor cell growth in vitroand in vivo. Ciardiello, F. et al. Inhibition of CRIPTO expression andtumorigenicity in human colon cancer cells by antisense RNA andoligodeoxynucleotides. Oncogene. 9(1):291-8, 1994 Jan. which are bothhereby incorporated herein by reference including all references citedtherein which are also hereby incorporated herein by reference.

[0275] Many carcinoma cells secrete transforming growth factor alpha. A23 nucleotide antisense oligonucleotide to TGF alpha mRNA inhibited bothDNA synthesis an proliferation of colorectal cancer cells. Sizeland, AM, Burgess, A W. Antisense transforming growth factor alphaoligonucleotides inhibit autocrine stimulated proliferation of a coloncarcinoma cell line. Molecular Biology of the Cell. 3(11):1235-43, 1992Nov. which is hereby incorporated herein by reference including allreferences cited therein which are also hereby incorporated herein byreference.

[0276] Human colorectal tumors have been identified with deletions ofchromosome 1p. It appears that a portion of this chromosome, 1p36-34,contains a tumor suppressor gene that regulates the expression of MYC.Tanaka, K, et al. Suppression of tumorigenicity in human colon carcinomacells by introduction of normal chromosome 1p36 region. Oncogene.8(8):2253-8, 1993 Aug, which is hereby incorporated herein by referenceincluding all references cited therein which are also herebyincorporated herein by reference.

[0277] Antisense compositions including oligonucleotides, derivativesand analogs thereof, conjugation protocols, and antisense strategies forinhibition of transcription and translation are generally described in:Antisense Research and Applications, Crooke, S. and B. Lebleu, eds. CRCPress, Inc. Boca Raton Fla. 1993; Nucleic Acids in Chemistry and BiologyBlackburn, G. and M. J. Gait, eds. IRL Press at Oxford University Press,Inc. New York 1990; and Oligonucleotides and Analogues: A PracticalApproach Eckstein, F. ed., IRL Press at Oxford University Press, Inc.New Yoprk 1991; which are each hereby incorporated herein by referenceincluding all references cited therein which are hereby incorporatedherein by reference.

[0278] The antisense molecules of the present invention comprise asequence complementary to a fragment of a colorectal cancer gene. SeeUllrich et al., EMBO J., 1986, 5:2503, which is hereby incorporatedherein by reference. Contemplated by this definition are fragments ofoligos within the coding sequence of colorectal cancer genes.

[0279] Antisense compositions which can make up an active moiety inconjugated compounds of the invention include oligonucleotides formed ofhomopyrimidines can recognize local stretches of homopurines in the DNAdouble helix and bind to them in the major groove to form a triplehelix. See: Helen, C and Toulme, J J. Specific regulation of geneexpression by antisense, sense, and antigene nucleic acids. Biochem.Biophys Acta, 1049:99-125, 1990 which is hereby incorporated herein byreference including all references cited therein which are herebyincorporated herein by reference. Formation of the triple helix wouldinterrupt the ability of the specific gene to undergo transcription byRNA polymerase. Triple helix formation using myc-specificoligonucleotides has been observed. See: Cooney, M, et al. Science241:456-459 which is hereby incorporated herein by reference includingall references cited therein which are hereby incorporated herein byreference.

[0280] Antisense oligonucleotides of DNA or RNA complementary tosequences at the boundary between introns and exons can be employed toprevent the maturation of newly-generated nuclear RNA transcripts ofspecific genes into mRNA for transcription.

[0281] Antisense RNA complimentary to specific genes can hybridize withthe mRNA for tat gene and prevent its translation. Antisense RNA can beprovided to the cell as “ready-to-use” RNA synthesized in vitro or as anantisense gene stably transfected into cells which will yield antisenseRNA upon transcription. Hybridization with mRNA results in degradationof the hybridized molecule by RNAse H and/or inhibition of the formationof translation complexes. Both result in a failure to produce theproduct of the original gene.

[0282] Antisense sequences of DNA or RNA can be delivered to cells.Several chemical modifications have been developed to prolong thestability and improve the function of these molecules withoutinterfering in their ability to recognize specific sequences. Theseinclude increasing their resistance to degradation by DNases, includingphosphotriesters, methylphosphonates, phosphorothioates, alpha-anomers,increasing their affinity for their s by covalent linkage to variousintercalating agents such as psoralens, and increasing uptake by cellsby conjugation to various groups including polylysine. These moleculesrecognize specific sequences encoded in mRNA and their hybridizationprevents translation of and increases the degradation of these messages.

[0283] Conjugated compositions of the invention provide a specific andeffective means for terminating the expression of genes which causeneoplastic transformation. CRCA-1 translation products undergoligand-induced endocytosis and can deliver conjugated compounds to thecytoplasm of cells when the CRCA-1 translation product binding moietybinds to an ST receptor on a colon cell. The unique localization ofthese receptors and their ability to undergo endocytosis make themexcellent candidates for targeting therapeutics to these tumors.

[0284] CRCA-1 translation product binding moieties are conjugateddirectly to antisense compositions such as nucleic acids which areactive in inducing a response in colorectal tumor cells. For example,antisense oligonucleotides to MYC are conjugated directly to ananti-CRCA-1 translation product antibody. This has been performedemploying peptides that bind to the CD4 receptor. See: Cohen, J S, ed.Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression. Topicsin Molecular and Structural Biology. CRC Press, Inc., Boca Raton, 1989.which is hereby incorporated herein by reference including allreferences cited therein which are hereby incorporated herein byreference. The precise backbone and its synthesis is not specified andcan be selected from well-established techniques. Synthesis wouldinvolve either chemical conjugation or direct synthesis of the chimericmolecule by solid phase synthesis employing FMOC chemistry. See:Haralambidis, J, et al. (1987) Tetrahedron Lett. 28:5199-5202, which ishereby incorporated herein by reference including all references citedtherein which are hereby incorporated herein by reference.Alternatively, the peptide-nucleic acid conjugate may be synthesizeddirectly by solid phase synthesis as a peptide-peptide nucleic acidchimera by solid phase synthesis. Nielsen, P E, et al. (1994)Sequence-specific transcription arrest by peptide nucleic acid bound tothe DNA template strand. Gene 149:139-145, which is hereby incorporatedherein by reference including all references cited therein which arehereby incorporated herein by reference.

[0285] In some embodiments, polylysine can be complexed to conjugatedcompositions of the invention in a non-covalent fashion to nucleic acidsand used to enhance delivery of these molecules to the cytoplasm ofcells. In addition, peptides and proteins can be conjugated topolylysine in a covalent fashion and this conjugate complexed withnucleic acids in a non-covalent fashion to further enhance thespecificity and efficiency of uptake of the nucleic acids into cells.Thus, CRCA-1 translation product ligand is conjugated chemically topolylysine by established techniques. The polylysine-CRCA-1 translationproduct ligand conjugate may be complexed with nucleic acids of choice.Thus, polylysine-orosomucoid conjugates were employed to specificallyplasmids containing genes to be expressed to hepatoma cells expressingthe orosomucoid receptor. This approach can be used to delivery wholegenes, or oligonucleotides. Thus, it has the potential to terminate theexpression of an undesired gene (eg. MYC, ras) or replace the functionof a lost or deleted gene (eg. hMSH2, hMLH1, hPMS1, and hPMS2).

[0286] According to a preferred embodiment, Myc serves as a gene whoseexpression is inhibited by an antisense molecule within a conjugatedcomposition. Many, if not most, colorectal tumor cells overexpress MYC,a gene involved in mediating proliferation. Decreasing the proliferationof colorectal tumor cells is attained by employing antisenseoligonucleotides complimentary to MYC to hybridize with the mRNA forthis protein, resulting in the degradation of this message and adramatic reduction in the production of MYC. CRCA-1 translation productbinding moieties are used to deliver a 15-based antisenseoligonucleotide to myc complementary to the translation initiationregion of exon II. This construct was active in inhibiting theexpression of MYC when it was incubated with colorectal cancer cells.The 15-base antisense oligonucleotide to MYC is synthesized as reportedin Collins, J F, Herman, P, Schuch, C, Bagby G C, Jr. Journal ofClinical Investigation. 89(5):1523-7, 1992 May. In some embodiments, theconjugated composition is conjugated to polylysine as reportedpreviously. Wu, G Y, and Wu, C H. (1988) Evidence for ed gene deliveryto Hep G2 hepatoma cells in vitro. Biochem. 27:887-892 which isincorporated herein by reference.

[0287] Conjugated compositions may be synthesized as a chimeric moleculedirectly by solid phase synthesis pmolar to nanomolar concentrations forthis conjugate suppress MYC synthesis in colorectal cancer cells invitro.

[0288] Antisense molecules are preferably hybridize to, i.e. arecomplementary to, a nucleotide sequence that is 5-50 nucleotides inlength, more preferably 5-25 nucleotides and in some embodiments 10-15nucleotides.

[0289] In addition, mismatches within the sequences identified above,which achieve the methods of the invention, such that the mismatchedsequences are substantially complementary to the colorectal cancer genesequences are also considered within the scope of the disclosure.Mismatches which permit substantial complementarity to the colorectalcancer gene sequences will be known to those of skill in the art oncearmed with the present disclosure. The oligos may also be unmodified ormodified.

[0290] Therapeutic compositions and methods may be used to combatcolorectal cancer in cases where the cancer is localized and/ormetastasized. Individuals are administered a therapeutically effectiveamount of conjugated compound. A therapeutically effective amount is anamount which is effective to cause a cytotoxic or cytostatic effect onmetastasized colorectal cancer cells without causing lethal side effectson the individual. An individual who has been administered atherapeutically effective amount of a conjugated composition has aincreased chance of eliminating colon cancer as compared to the risk hadthe individual not received the therapeutically effective amount.

[0291] To treat localized colorectal cancer, a therapeutically effectiveamount of a conjugated compound is administered such that it will comeinto contact with the localized tumor within the colon. Thus, theconjugated compound is administered orally or rectally. In cases whereconjugated compounds are orally administered, they are preferablyenteric coated or otherwise formulated to avoid degradation by stomachacids. Enteric formulations are described in U.S. Pat. No. 4,601,896,U.S. Pat. No. 4,729,893, U.S. Pat. No. 4,849,227, U.S. Pat. No.5,271,961,U.S. Pat. No. 5,350,741, and U.S. Pat. No. 5,399,347, whichare each hereby incorporated herein by reference. Oral and rectalformulation are taught in Remington's Pharmaceutical Sciences, 18thEdition, 1990, Mack Publishing Co., Easton Pa. which is incorporatedherein by reference. Alternative embodiments include sustained releaseformulations and implant devices which provide continuous delivery ofconjugated compositions to the colon.

[0292] The pharmaceutical compositions according to the presentinvention may be administered as either a single dose or in multipledoses. The pharmaceutical compositions of the present invention may beadministered either as individual therapeutic agents or in combinationwith other therapeutic agents. The treatments of the present inventionmay be combined with conventional therapies, which may be administeredsequentially or simultaneously.

[0293] The present invention is directed to a method of deliveringantisense compounds to colon cells and inhibiting expression ofcolorectal cancer genes in mammals. The methods comprise administeringto a mammal an effective amount of a conjugated composition whichcomprises a CRCA-1 translation product binding moiety conjugated to anantisense oligonucleotide having a sequence which is complementary to aregion of DNA or mRNA of a colorectal cancer gene.

[0294] The conjugated compounds may be administering to mammals in amixture with a pharmaceutically-acceptable carrier, selected with regardto the intended route of administration and the standard pharmaceuticalpractice. Dosages will be set with regard to weight, and clinicalcondition of the patient. The conjugated compositions of the presentinvention will be administered for a time sufficient for the mammals tobe free of undifferentiated cells and/or cells having an abnormalphenotype. In therapeutic methods treatment extends for a timesufficient to inhibit transformed cells from proliferating andconjugated compositions may be administered in conjunction with otherchemotherapeutic agents to manage and combat the patient's cancer.

[0295] The conjugated compounds of the invention may be employed in themethod of the invention singly or in combination with other compounds.The amount to be administered will also depend on such factors as theage, weight, and clinical condition of the patient. See Gennaro,Alfonso, ed., Remington's Pharmaceutical Sciences, 18th Edition, 1990,Mack Publishing Co., Easton Pa.

[0296] Prophylactic compositions and methods may be used to prevent theorigin of colorectal cancer. In particular, conjugated compounds may beadministered to an individual is suspected of being susceptible tocolorectal cancer. Using genotyping techniques, the specific nature ofan individuals susceptibility may be identified. That is, it may bepossible to determine what cancer gene will be associated withcolorectal cancer in an individual. For example, defects in the APC geneand kits for diagnosing the same are disclosed in U.S. Pat. No.5,352,775 which is hereby incorporated herein by reference. Similarly,defects in the MCC gene and kits for diagnosing the same are disclosedin U.S. Pat. No. 5,330,892 which is hereby incorporated herein byreference. In prophylactic methods, treatment extends continuously orsporadically from time to time for a time sufficient to inhibittransformation.

[0297] To prevent colorectal cancer, a prophylactically effective amountof a conjugated compound is administered such that it will come intocontact with and incorporated by normal colon cells. Thus, theconjugated compound is administered orally or rectally. In cases whereconjugated compounds are orally administered, they are preferablyenteric coated or otherwise formulated to avoid degradation by stomachacids as described above.

[0298] A prophylactically effective amount is an amount which iseffective to prevent the initiation of transformation of colon cancer incells. An individual who has been administered a prophylacticallyeffective amount of a conjugated composition has a reduced risk ofdevelopment of colon cancer as compared to the risk had the individualnot received the prophylactically effective.

[0299] Therapeutic and Prophylactic Vaccines

[0300] The invention relates to prophylactic and therapeutic vaccinesfor protecting individuals against metastatic colorectal cancer and fortreating individuals who are suffering from metastatic colorectalcancer.

[0301] According to the present invention, one or more of the CRCA-1translation products serves as a target against which a protective andtherapeutic immune response can be induced. Specifically, vaccines areprovided which induce an immune response against a CRCA-1 translationproduct. The vaccines of the invention include, but are not limited to,the following vaccine technologies:

[0302] 1) DNA vaccines, i.e. vaccines in which DNA that encodes at leastan epitope from a CRCA-1 translation product that is not present on STreceptor protein is administered to an individual's cells where theepitope is expressed and serves as a target for an immune response;

[0303] 2) infectious vector mediated vaccines such as recombinantadenovirus, vaccinia, Salmonella, and BCG wherein the vector carriesgenetic information that encodes at least an epitope from a CRCA-1translation product that is not present on ST receptor protein such thatwhen the infectious vector is administered to an individual, the epitopeis expressed and serves as a target for an immune response;

[0304] 3) killed or inactivated vaccines which a) comprise either killedcells or inactivated viral particles that display at least an epitopefrom a CRCA-1 translation product that is not present on ST receptorprotein and b) when administered to an individual serves as a target foran immune response;

[0305] 3) haptenized killed or inactivated vaccines which a) compriseeither killed cells or inactivated viral particles that display at leastan epitope from a CRCA-1 translation product that is not present on STreceptor protein, b) are haptenized to be more immunogenic and c) whenadministered to an individual serves as a target for an immune response;

[0306] 4) subunit vaccines which are vaccines that include proteinmolecules that include at least an epitope from a CRCA-1 translationproduct that is not present on ST receptor protein; and

[0307] 5) haptenized subunit vaccines which are vaccines that a) includeprotein molecules that include at least an epitope from a CRCA-1translation product that is not present on ST receptor protein and b)are haptenized to be more immunogenic.

[0308] The present invention relates to administering to an individual aprotein or nucleic acid molecule that comprises or encodes,respectively, an immunogenic epitope against which an therapeutic andprophylactic immune response can be induced. Such epitopes are generallyat least 6-8 amino acids in length. The vaccines of the inventiontherefore comprise proteins which are at least, or nucleic acids whichencode at least, 6-8 amino acids in length from one or more CRCA-1translation products that is not present on ST receptor protein. Thevaccines of the invention may comprise proteins which are at least, ornucleic acids which encode at least 10 to about 1000 amino acids inlength. The vaccines of the invention may comprise proteins which are atleast, or nucleic acids which encode at least, about 25 to about 500amino acids in length. The vaccines of the invention may compriseproteins which are at least, or nucleic acids which encode at least,about 50 to about 400 amino acids in length. The vaccines of theinvention may comprise proteins which are at least, or nucleic acidswhich encode at least, about 100 to about 300 amino acids in length.

[0309] The present invention relates to compositions for and methods oftreating individuals who are known to have metastasized colorectalcancer. Metastasized colorectal cancer may be diagnosed by those havingordinary skill in the art using art accepted clinical and laboratorypathology protocols and/or those described in U.S. Ser. No. 08/141,892filed on Oct. 26, 1993, U.S. Ser. No. 08/305,056 filed on Sep. 13, 1994,and PCT Application Serial Number PCT/US94/12232 filed Oct. 26, 1994.The present invention provides an immunotherapeutic vaccine useful totreat individuals who have been diagnosed as suffering from metastasizedcolorectal cancer. The immunotherapeutic vaccines of the presentinvention may be administered in combination with other therapiesincluding, but not limited to those described in U.S. Ser. No.08/141,892 filed on Oct. 26, 1993, U.S. Ser. No. 08/305,056 filed onSep. 13, 1994, and PCT Application Serial Number PCT/US94/12232 filedOct. 26, 1994.

[0310] The present invention relates to compositions for and methods ofpreventing metastatic colorectal cancer in individual is suspected ofbeing susceptible to metastasized colorectal cancer. Such individualsinclude those whose family medical history indicates above averageincidence of colorectal cancer among family members and/or those whohave already developed colorectal cancer and have been effectivelytreated who therefore face a risk of relapse and recurrence. Suchindividuals include those which have been diagnosed as having colorectalcancer including localized only or localized and metastasized colorectalcancer which has been resected or otherwise treated. Such individualsalso include those with an elevated risk as ascertained by geneticevaluation. For example, individuals with APC mutations can beidentified following the U.S. Pat. No. 5,352,775 issued Oct. 4, 1992 toAlbertsen et al., which is incorporated herein by reference.Furthermore, such individuals include: those suffering from inflammatorybowel disease, particularly those with ulcerative colitis; those withcolonic polyps; those with familial adenomatous polyposis, a heritablemutation predisposing patients to develop large numbers of intestinalpolyps; those with Peutz-Jeghers syndrome; those with hereditarynonpolyposis coli, a heritable mutation which predisposes people todevelop colon carcinoma; those with Turcot syndrome-colon carcinoma inconjunction with independent tumors of the central nervous system; andindividuals engaging in rectal intercourse. The vaccines of the presentinvention may be to susceptible individuals prophylactically to preventand combat colorectal cancer metastasis.

[0311] The invention relates to compositions which are the activecomponents of such vaccines or required to make the active components,to methods of making such compositions including the active components,and to methods of making and using vaccines.

[0312] The nucleotide sequence of the CRCA-1 transcript is set forth asSEQ ID NO:1 and the amino acid sequences of the various translationproducts are set forth in SEQ ID NOs:2-81. The present invention relatesto isolated fragments of the CRCA-1 transcript that encode specificCRCA-1 translation products.

[0313] The present invention relates to recombinant vectors, includingexpression vectors, that comprise the CRCA-1 transcript or a fragmentthereof. The present invention relates to recombinant vectors, inlcudingexpression vectors that comprise nucleotide sequences that encode aCRCA-1 translation product or a functional fragment thereof.

[0314] The present invention relates to host cells which comprise suchvectors and to methods of making CRCA-1 translation products using suchrecombinant cells.

[0315] The present invention relates to the isolated CRCA-1 transcriptand to the isolated CRCA-1 translation products and to isolatedantibodies specific for such products and to hybridomas which producesuch antibodies.

[0316] The present invention relates to the isolated CRCA-1 translationproducts and functional fragments thereof. Accordingly, some aspects ofthe invention relate to isolated proteins that comprise at least oneepitope of a CRCA-1 translation product.

[0317] Some aspects of the invention relate to the above describedisolated proteins which are haptenized to render them more immunogenic.That is, some aspects of the invention relate to haptenized proteinsthat comprise at least one CRCA-1 translation product epitope.

[0318] Accordingly, some aspects of the invention relate to isolatednucleic acid molecules that encode proteins that comprise at least oneCRCA-1 translation product epitope.

[0319] Naked DNA vaccines are described in PCT/US90/01515, which isincorporated herein by reference. Others teach the use of liposomemediated DNA transfer, DNA delivery using microprojectiles (U.S. Pat.No. 4,945,050 issued Jul. 31, 1990 to Sanford et al., which isincorporated herein by reference), and DNA delivery usingelectroporation. In each case, the DNA may be plasmid DNA that isproduced in bacteria, isolated and administered to the animal to betreated. The plasmid DNA molecules are taken up by the cells of theanimal where the sequences that encode the protein of interest areexpressed. The protein thus produced provides a therapeutic orprophylactic effect on the animal.

[0320] The use of vectors including viral vectors and other means ofdelivering nucleic acid molecules to cells of an individual in order toproduce a therapeutic and/or prophylactic immunological effect on theindividual are similarly well known. Recombinant vaccines that employvaccinia vectors are, for example, disclosed in U.S. Pat. No. 5,017,487issued May 21, 1991 to Stunnenberg et al. which is incorporated hereinby reference.

[0321] In some cases, tumor cells from the patient are killed orinactivated and administered as a vaccine product. Berd et al. May 1986Cancer Research 46:2572-2577 and Berd et al. May 1991 Cancer Research51:2731-2734, which are incorporated herein by reference, describes thepreparation and use of tumor cell based vaccine products. According tosome aspects of the present invention, the methods and techniquesdescribed in Berd et al. are adapted by using colorectal cancer cellsinstead of melanoma cells.

[0322] The manufacture and use of isolated translation products andfragments thereof useful for example as laboratory reagents orcomponents of subunit vaccines are well known. One having ordinary skillin the art can isolate the CRCA-1 transcript or the specific portionthereof that encodes a CRCA-1 translation product or a fragment thereof.Once isolated, the nucleic acid molecule can be inserted it into anexpression vector using standard techniques and readily availablestarting materials.

[0323] The recombinant expression vector that comprises a nucleotidesequence that encodes the nucleic acid molecule that encodes a CRCA-1translation product or a fragment thereof or a protein that comprisesthe CRCA-1 translation product or a fragment thereof. The recombinantexpression vectors of the invention are useful for transforming hosts toprepare recombinant expression systems for preparing the isolatedproteins of the invention.

[0324] The present invention relates to a host cell that comprises therecombinant expression vector that includes a nucleotide sequence thatencodes one or more CRCA-1 translation products or a fragment thereof ora protein that comprises one or more CRCA-1 translation products or afragment thereof. Host cells for use in well known recombinantexpression systems for production of proteins are well known and readilyavailable. Examples of host cells include bacteria cells such as E.coli, yeast cells such as S. cerevisiae, insect cells such as S.frugiperda, non-human mammalian tissue culture cells chinese hamsterovary (CHO) cells and human tissue culture cells such as HeLa cells.

[0325] The present invention relates to a transgenic non-human mammalthat comprises the recombinant expression vector that comprises anucleic acid sequence that encodes the proteins of the invention.Transgenic non-human mammals useful to produce recombinant proteins arewell known as are the expression vectors necessary and the techniquesfor generating transgenic animals. Generally, the transgenic animalcomprises a recombinant expression vector in which the nucleotidesequence that encodes one or more CRCA-1 translation products or afragment thereof or a protein that comprises the one or more CRCA-1translation products or a fragment thereof operably linked to a mammarycell specific promoter whereby the coding sequence is only expressed inmammary cells and the recombinant protein so expressed is recovered fromthe animal's milk.

[0326] In some embodiments, for example, one having ordinary skill inthe art can, using well known techniques, insert such DNA molecules intoa commercially available expression vector for use in well knownexpression systems such as those described herein.

[0327] The expression vector including the DNA that encodes a CRCA-1translation product or a functional fragment thereof or a protein thatcomprises a CRCA-1 translation product or a functional fragment thereofis used to transform the compatible host which is then cultured andmaintained under conditions wherein expression of the foreign DNA takesplace. The protein of the present invention thus produced is recoveredfrom the culture, either by lysing the cells or from the culture mediumas appropriate and known to those in the art. The methods of purifyingthe CRCA-1 translation products or a fragment thereof or a protein thatcomprises the same using antibodies which specifically bind to theprotein are well known. Antibodies which specifically bind to aparticular protein may be used to purify the protein from naturalsources using well known techniques and readily available startingmaterials. Such antibodies may also be used to purify the protein frommaterial present when producing the protein by recombinant DNAmethodology. The present invention relates to antibodies that bind to anepitope which is present on one or more CRCA-1 translation products or afragment thereof or a protein that comprises the same. Antibodies thatbind to an epitope which is present on the CRCA-1 translation productare useful to isolate and purify the protein from both natural sourcesor recombinant expression systems using well known techniques such asaffinity chromatography. Immunoaffinity techniques generally aredescribed in Waldman et al. 1991 Methods of Enzymol. 195:391-396, whichis incorporated herein by reference. Antibodies are useful to detect thepresence of such protein in a sample and to determine if cells areexpressing the protein. The production of antibodies and the proteinstructures of complete, intact antibodies, Fab fragments and F(ab)₂fragments and the organization of the genetic sequences that encode suchmolecules are well known and are described, for example, in Harlow, E.and D. Lane (1988) ANTIBODIES: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. which is incorporated herein byreference.

[0328] In some embodiments of the invention, transgenic non-humananimals are generated. The transgenic animals according to the inventioncontain nculeotides that encode one or more CRCA-1 translation productsor a fragment thereof or a protein that comprises the same under theregulatory control of a mammary specific promoter. One having ordinaryskill in the art using standard techniques, such as those taught in U.S.Pat. No. 4,873,191 issued Oct. 10, 1989 to Wagner and U.S. Pat. No.4,736,866 issued Apr. 12, 1988 to Leder, both of which are incorporatedherein by reference, can produce transgenic animals which produce one ormore CRCA-1 translation products or a fragment thereof or a protein thatcomprises the same. Preferred animals are goats and rodents,particularly rats and mice.

[0329] In addition to producing these proteins by recombinanttechniques, automated peptide synthesizers may also be employed toproduce one or more CRCA-1 translation products or a fragment thereof ora fragment thereof or a protein that comprises the same. Such techniquesare well known to those having ordinary skill in the art and are usefulif derivatives which have substitutions not provided for in DNA-encodedprotein production.

[0330] In some embodiments, the protein that makes up a subunit vaccineor the cells or particles of a killed or inactivated vaccine may behaptenized to increase immunogenicity. In some cases, the haptenizationis the conjugation of a larger molecular structure to one or more CRCA-1translation products or a fragment thereof or a protein that comprisesthe same. In some cases, tumor cells from the patient are killed andhaptenized as a means to make an effective vaccine product. In cases inwhich other cells, such as bacteria or eukaryotic cells which areprovided with the genetic information to make and display a CRCA-1translation product or a fragment thereof or a protein that comprisesthe same, are killed and used as the active vaccine component, suchcells are haptenized to increase immunogenicity. Haptenization is wellknown and can be readily performed.

[0331] Methods of haptenizing cells generally and tumor cells inparticular are described in Berd et al. May 1986 Cancer Research46:2572-2577 and Berd et al. May 1991 Cancer Research 51:2731-2734,which are incorporated herein by reference. Additional haptenizationprotocols are disclosed in Miller et al. 1976 J. Immunol.117(5:1):1591-1526.

[0332] Haptenization compositions and methods which may be adapted to beused to prepare haptenized CRCA-1 immunogens according to the presentinvention include those described in the following U.S. Patents whichare each incorporated herein by reference: U.S. Pat. No. 5,037,645issued Aug. 6, 1991 to Strahilevitz; U.S. Pat. No. 5,112,606 issued May12, 1992 to Shiosaka et al.; U.S. Pat. No. 4,526,716 issued Jul. 2, 1985to Stevens; U.S. Pat. No. 4,329,281 issued May 11, 1982 to Christensonet al.; and U.S. Pat. No. 4,022,878 issued May 10, 1977 to Gross.Peptide vaccines and methods of enhancing immunogenicity of peptideswhich may be adapted to modify CRCA-1 immunogens of the invention arealso described in Francis et al. 1989 Methods of Enzymol. 178:659-676,which is incorporated herein by reference. Sad et al. 1992 Immunolology76:599-603, which is incorporated herein by reference, teaches methodsof making immunotherapeutic vaccines by conjugating gonadotropinreleasing hormone to diphtheria toxoid. CRCA-1 immunogens may besimilarly conjugated to produce an immunotherapeutic vaccine of thepresent invention. MacLean et al. 1993 Cancer Immunol. Immunother.36:215-222, which is incorporated herein by reference, describesconjugation methodologies for producing immunotherapeutic vaccines whichmay be adaptable to produce an immunotherapeutic vaccine of the presentinvention. The hapten is keyhole limpet hemocyanin which may beconjugated to a CRCA-1 immunogen.

[0333] Vaccines according to some aspects of the invention comprise apharmaceutically acceptable carrier in combination with a CRCA-1immunogen. Pharmaceutical formulations are well known and pharmaceuticalcompositions comprising such proteins may be routinely formulated by onehaving ordinary skill in the art. Suitable pharmaceutical carriers aredescribed in Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field, which is incorporated herein by reference.The present invention relates to an injectable pharmaceuticalcomposition that comprises a pharmaceutically acceptable carrier and aCRCA-1 immunogen. The CRCA-1 immunogen is preferably sterile andcombined with a sterile pharmaceutical carrier.

[0334] In some embodiments, for example, one or more CRCA-1 translationproducts or a fragment thereof or a fragment thereof or a protein thatcomprises the same can be formulated as a solution, suspension, emulsionor lyophilized powder in association with a pharmaceutically acceptablevehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 5% human serum albumin. Liposomes and nonaqueousvehicles such as fixed oils may also be used. The vehicle or lyophilizedpowder may contain additives that maintain isotonicity (e.g., sodiumchloride, mannitol) and chemical stability (e.g., buffers andpreservatives). The formulation is sterilized by commonly usedtechniques.

[0335] An injectable composition may comprise the CRCA-1 immunogen in adiluting agent such as, for example, sterile water,electrolytes/dextrose, fatty oils of vegetable origin, fatty esters, orpolyols, such as propylene glycol and polyethylene glycol. Theinjectable must be sterile and free of pyrogens.

[0336] The vaccines of the present invention may be administered by anymeans that enables the immunogenic agent to be presented to the body'simmune system for recognition and induction of an immunogenic response.Pharmaceutical compositions may be administered parenterally, i.e.,intravenous, subcutaneous, intramuscular.

[0337] Dosage varies depending upon known factors such as thepharmacodynamic characteristics of the particular agent, and its modeand route of administration; age, health, and weight of the recipient;nature and extent of symptoms, kind of concurrent treatment, frequencyof treatment, and the effect desired. An amount of immunogen isdelivered to induce a protective or therapeutically effective immuneresponse. Those having ordinary skill in the art can readily determinethe range and optimal dosage by routine methods.

[0338] The following examples are illustrative but are not meant to belimiting of the present invention.

EXAMPLES Example 1

[0339] As stated above, a CRCA-1 translation product binding moiety is aCRCA-1 translation product ligand that may be an antibody, a protein, apolypeptide, a peptide or a non-peptide. Peptides and non-peptide CCK Areceptor specific ligands may be identified using well known technology.

[0340] Over the past 10 years, it has become recognized that thespecific high-affinity interaction of a receptor and a ligand, forexample a CRCA-1 translation product and an anti-CRCA-1 translationproduct antibody, has its basis in the 3-dimensional conformationalspace of the ligand and the complimentary 3-dimensional configuration ofthe region of the molecule involved in ligand binding. In addition, ithas become recognized that various arrays of naturally-occurring aminoacids, non-natural amino acids, and organic molecules can be organizedin configurations that are unrelated to the natural ligands in theirlinear structure, but resemble the 3-dimensional structure of thenatural ligands in conformational space and, thus, are recognized byreceptors with high affinity and specificity. Furthermore, techniqueshave been described in the literature that permit one of ordinary skillin the art to generate large libraries of these arrays of natural aminoacids, non-natural amino acids and organic compounds to prospectivelyidentify individual compounds that interact with receptors with highaffinity and specificity which are unrelated to the native ligand ofthat receptor. Thus, it is a relatively straightforward task for one ofordinary skill in the art to identify arrays of naturally occurringamino acids, non-natural amino acids, or organic compounds which canbind specifically and tightly to the CRCA-1 translation product, whichbear no structural relationship to an anti-CRCA-1 translation productantibody.

[0341] To identify CRCA-1 translation product ligands that are peptides,those having ordinary skill in the art can use any of the well knownmethodologies for screening random peptide libraries in order toidentify peptides which bind to the CRCA-1 translation product. In themost basic of methodologies, the peptides which bind to the target areisolated and sequenced. In some methodologies, each random peptide islinked to a nucleic acid molecule which includes the coding sequence forthat particular random peptide. The random peptides, each with anattached coding sequence, are contacted with a CRCA-1 translationproduct and the peptides which are unbound to the CRCA-1 translationproduct are removed. The nucleic acid molecule which includes the codingsequence of the peptide that binds to the CRCA-1 translation product canthen be used to determine the amino acid sequence of the peptide as wellas produce large quantities of the peptide. It is also possible toproduce peptide libraries on solid supports where the spatial locationon the support corresponds to a specific synthesis and thereforespecific peptide. Such methods often use photolithography-like steps tocreate diverse peptide libraries on solid supports in which the spatialaddress on the support allows for the determination of the sequence.

[0342] The production of organic compound libraries on solid supportsmay also be used to produce combinatorial libraries of non-peptidecompounds such as oligonucleotides and sugars, for example. As in thecase of peptide libraries on solid supports, the spatial location on thesupport corresponds to a specific synthesis and therefore specificcompound. Such methods often use photolithography-like steps to creatediverse compound libraries on solid supports in which the spatialaddress on the support allows for the determination of the synthesisscheme which produced the compound. Once the synthesis scheme isidentified, the structure of the compound can become known.

[0343] Gallop et al. 1994 J. Medicinal Chemistry 37:1233, which isincorporated herein by reference, provides a review of several of thevarious methodologies of screening random peptide libraries andidentifying peptides from such libraries which bind to target proteins.Following these teachings, CRCA-1 translation product specific ligandsthat are peptides and that are useful as CRCA-1 translation productspecific binding moieties may be identified by those having ordinaryskill in the art.

[0344] Peptides and proteins displayed on phage particles are describedin Gallop et al. Supra. Random arrays of nucleic acids can be insertedinto genes encoding surface proteins of bacteriophage which are employedto infect bacteria, yielding phage expressing the peptides encoded bythe random array of nucleotides on their surface. These phage displayingthe peptide can be employed to determine whether those peptides can bindto specific proteins, receptors, antibodies, etc. The identity of thepeptide can be determined by sequencing the recombinant DNA from thephage expressing the peptide. This approach has the potential to yieldvast arrays of peptides in a library (up to 10⁹ unique peptides). Thistechnique has been employed to identify novel binding peptides to thefibrinogen receptor on platelets, which bear no sequence homology to thenatural occurring ligands of this receptor (Smith et al., 1993 Gene128:37, which is incorporated herein by reference). Similarly, thistechnique has been applied to identify peptides which bind to the MHCclass II receptor (Hammer et al., 1993 Cell 74:197, which isincorporated herein by reference) and the chaperonin receptor(Blond-Elguindi et al., 1993 Cell 75:717, which is incorporated hereinby reference).

[0345] Peptides displayed on plasmids are described in Gallop et al.Supra. In this approach, the random oligonucleotides which encode thelibrary of peptides can be expressed on a specific plasmid whoseexpression is under the control of a specific promoter, such as the lacoperon. The peptides are expressed as fusion proteins coupled to the LacI protein, under the control of the lac operon. The fusion proteinspecifically binds to the lac operator on the plasmid and so the randompeptide is associated with the specific DNA element that encodes it. Inthis way, the sequence of the peptide can be deduced, by PCR of the DNAassociated with the fusion protein. These proteins can be screened insolution phase to determine whether they bind to specific receptors.Employing this approach, novel substrates have been identified forspecific enzymes (Schatz 1993).

[0346] A variation of the above technique, also described in Gallop etal. Supra, can be employed in which random oligonucleotides encodingpeptide libraries on plasmids can be expressed in cell-free systems. Inthis approach, a molecular DNA library can be constructed containing therandom array of oligonucleotides, which are then expressed in abacterial in vitro transcription/translation system. The identity of theligand is determined by purifying the complex of nascent chainpeptide/polysome containing the mRNA of interest on affinity resinscomposed of the receptor and then sequencing following amplificationwith RT-PCR. Employing this technique permits generation of largelibraries (up to 10¹¹ recombinants). Peptides which recognize antibodiesspecifically directed to dynorphin have been identified employing thistechnique (Cull et al., 1992 Proc. Natl. Acad. Sci. USA 89:1865, whichis incorporated herein by reference).

[0347] Libraries of peptides can be generated for screening against areceptor by chemical synthesis. For example, simultaneous preparation oflarge numbers of diverse peptides have been generated employing theapproach of multiple peptide synthesis as described in Gallop et al.Supra. In one application, random peptides are generated by standardsolid-phase Merrifield synthesis on polyacrylamide microtiter plates(multipin synthesis) which are subsequently screened for their abilityto compete with receptor binding in a standard competitive binding assay(Wang et al., 1993 Bioorg. Med. Chem. Lett. 3:447, which is incorporatedherein by reference). Indeed, this approach has been employed toidentify novel binding peptides to the substance P receptor (Wang et al.Supra). Similarly, peptide libraries can be constructed by multiplepeptide synthesis employing the “tea bag” method in which bags of solidsupport resin are sequentially incubated with various amino acids togenerate arrays of different peptides (Gallop et al. Supra). Employingthis approach, peptides which bind to the integrin receptor (Ruggeri etal., 1986 Proc. Natl. Acad. Sci. USA 83:5708, which is incorporatedherein by reference) and the neuropeptide Y receptor (Beck-Sickinger etal., 1990 Int. J. Peptide Protein Res. 36:522, which is incorporatedherein by reference) have been identified.

[0348] In general, the generation and utility of combinatorial librariesdepend on (1) a method to generate diverse arrays of building blocks,(2) a method for identifying members of the array that yield the desiredfunction, and (3) a method for deconvoluting the structure of thatmember. Several approaches to these constraints have been defined.

[0349] The following is a description of methods of library generationwhich can be used in procedures for identifying CRCA-1 translationproduct specific ligands according to the invention.

[0350] Modifications of the above approaches can be employed to generatelibraries of vast molecular diversity by connecting together members ofa set of chemical building blocks, such as amino acids, in all possiblecombinations (Gallop et al. Supra) In one approach, mixtures ofactivated monomers are coupled to a growing chain of amino acids on asolid support at each cycle. This is a multivalent synthetic system.

[0351] Also, split synthesis involves incubating the growing chain inindividual reactions containing only a single building block (Gallop etal. Supra). Following attachment, resin from all the reactions are mixedand apportioned into individual reactions for the next step of coupling.These approaches yield a stochastic collection of n^(x) differentpeptides for screening, where n is the number of building blocks and xis the number of cycles of reaction.

[0352] Alternatively, arrays of molecules can be generated in which oneor more positions contain known amino acids, while the remainder arerandom (Gallop et al. Supra). These yield a limited library which isscreened for members with the desired activity. These members areidentified, their structure determined, and the structure regeneratedwith another position containing defined amino acids and screened. Thisiterative approach ultimately yields peptides which are optimal forrecognizing the conformational binding pocket of a receptor.

[0353] In addition, arrays are not limited to amino acids formingpeptides, but can be extended to linear and nonlinear arrays of organicmolecules (Gordon et al., 1994 J. Medicinal Chemistry 37:1385, which isincorporated herein by reference). Indeed, employing this approach ofgenerating libraries of randomly arrayed inorganic building blocks,ligands which bound to 7-transmembrane receptors were identified(Zuckermann et al., 1994 J. Med. Chem. 37:2678, which is incorporatedherein by reference).

[0354] Libraries are currently being constructed which can be modifiedafter synthesis to alter the chemical side groups and bonds, to give“designer” arrays to test for their interaction with receptors (Ostereshet al., 1994 Proc. Natl. Acad. Sci. USA 91:11138, which is incorporatedherein by reference). This technique, generating “libraries fromlibraries”, was applied to the permethylation of a peptide library whichyielded compounds with selective antimicrobial activity against grampositive bacteria.

[0355] Libraries are also being constructed to express arrays ofpharmacological motifs, rather than specific structural arrays of aminoacids(Sepetov et al., 1995 Proc. Natl. Acad. Sci. USA 92:5426, which isincorporated herein by reference). This technique seeks to identifystructural motifs that have specific affinities for receptors, which canbe modified in further refinements employing libraries to definestructure-activity relationships. Employing this approach of searchingmotif libraries, generating “libraries of libraries”, reduces the numberof component members required for screening in the early phase oflibrary examination.

[0356] The following is a description of methods of identifying CRCA-1translation product specific ligands according to the invention fromlibraries of randomly generated molecules.

[0357] Components in the library which interact with receptors may beidentified by their binding to receptors immobilized on solid support(Gordon et al. Supra).

[0358] They may also be identified by their ability to compete withnative ligand for binding to cognate receptors in solution phase (Gordonet al. Supra).

[0359] Components may be identified by their binding to solublereceptors when those components are immobilized on solid supports(Gordon et al. Supra).

[0360] Once a member of a library which binds receptors has beenidentified, the structure of that member must be deconvoluted (deduced)in order to identify the structure and generate large quantities to workwith, or develop further analogs to study structure-activityrelationships. The following is a description of methods ofdeconvolution for deducing the structure of molecules identified aspotential CRCA-1 translation product specific ligands according to theinvention.

[0361] Peptide libraries may be expressed on the surface ofbacteriophage particles (Gallop et al. Supra). Once the peptideinteracting with the receptor has been identified, its structure can bededuced by isolating the DNA from the phage and determining its sequenceby PCR.

[0362] Libraries expressed on plasmids, under the control of the Lacoperon can be deconvoluted since these peptides are fused with the lac Iprotein which specifically interacts with the lac operon on the plasmidencoding the peptide (Gallop et al. Supra) The structure can be deducedby isolating that plasmid attached to the lac I protein and deducing thenucleotide and peptide sequence by PCR.

[0363] Libraries expressed on plasmids can also be expressed incell-free systems employing transcription/translation systems (Gallop etal. Supra). In this paradigm, the protein interacting with receptors isisolated with its attached ribosome and mRNA. The sequence of thepeptide is deduced by RT-PCR of the associated mRNA.

[0364] Library construction can be coupled with photolithography, sothat the structure of any member of the library can be deduced bydetermining its position within the substrate array (Gallop et al.Supra). This technique is termed positional addressability, since thestructural information can be deduced by the precise position of themember.

[0365] Members of a library can also be identified by tagging thelibrary with identifiable arrays of other molecules (Ohlmeyer et al.,1993 Proc. Natl. Acad. Sci. USA 90:10922, which is incorporated hereinby reference, and Gallop et al. Supra). This technique is a modificationof associating the peptide with the plasmid of phage encoding thesequence, described above. Some methods employ arrays of nucleotides toencode the sequential synthetic history of the peptide. Thus,nucleotides are attached to the growing peptide sequentially, and can bedecoded by PCR to yield the structure of the associated peptide.Alternatively, arrays of small organic molecules can be employed assequencable tags which encode the sequential synthetic history of thepeptide. Thus, nucleotides are attached to the growing peptidesequentially, and can be decoded by PCR to yield the structure of theassociated peptide. Alternatively, arrays of small organic molecules canbe employed as sequencable tags which encode the sequential synthetichistory of the library member.

[0366] Finally, the structure of a member of the library can be directlydetermined by amino acid sequence analysis.

[0367] The following patents, which are each incorporated herein byreference, describe methods of making random peptide or non-peptidelibraries and screening such libraries to identify compounds that bindto target proteins. As used in the present invention, CRCA-1 translationproduct can be the targets used to identify the peptide and non-peptideligands generated and screened as disclosed in the patents.

[0368] U.S. Pat. No. 5,270,170 issued to Schatz et al. on Dec. 14, 1993,and U.S. Pat. No. 5,338,665 issued to Schatz et al. on Aug. 16, 1994,which are both incorporated herein by reference, refer to peptidelibraries and screening methods which can be used to identify CRCA-1translation product ligands.

[0369] U.S. Pat. No. 5,395,750 issued to Dillon et al. on Mar. 7, 1995,which is incorporated herein by reference, refers to methods ofproducing proteins which bind to predetermined antigens. Such methodscan be used to produce CRCA-1 translation product ligands.

[0370] U.S. Pat. No. 5,223,409 issued to Ladner et al. on Jun. 29, 1993,which is incorporated herein by reference, refers to the directedevolution to novel binding proteins. Such proteins may be produced andscreened as disclosed therein to identify CRCA-1 translation productligands.

[0371] U.S. Pat. No. 5,366,862 issued to Venton et al. on Nov. 22, 1994,which is incorporated herein by reference, refers to methods forgenerating and screening useful peptides. The methods herein describedcan be used to identify CRCA-1 translation product ligands.

[0372] U.S. Pat. No. 5,340,474 issued to Kauvar on Aug. 23, 1994 as wellas U.S. Pat. No. 5,133,866, U.S. Pat. No. 4,963,263 and U.S. Pat. No.5,217,869, which are each incorporated herein by reference, can be usedto identify CRCA-1 translation product ligands.

[0373] U.S. Pat. No. 5,405,783 issued to Pirrung et al. on Apr. 11,1995, which is incorporated herein by reference, refers to large scalephotolithographic solid phase synthesis of an array of polymers. Theteachings therein can be used to identify CRCA-1 translation productligands.

[0374] U.S. Pat. No. 5,143,854 issued to Pirrung et al. on Sep. 1, 1992,which is incorporated herein by reference, refers to a large scalephotolithographic solid phase synthesis of polypeptides and receptorbinding screening thereof.

[0375] U.S. Pat. No. 5,384,261 issued to Winkler et al. on Jan. 24,1995, which is incorporated herein by reference, refers to very largescale immobilized polymer synthesis using mechanically directed flowpatterns. Such methods are useful to identify CRCA-1 translation productligands.

[0376] U.S. Pat. No. 5,221,736 issued to Coolidge et al. on Jun. 22,1993, which is incorporated herein by reference, refers to sequentialpeptide and oligonucleotide synthesis using immunoaffinity techniques.Such techniques may be used to identify CRCA-1 translation productligands.

[0377] U.S. Pat. No. 5,412,087 issued to McGall et al. on May 2, 1995,which is incorporated herein by reference, refers to spatiallyaddressable immobilization of oligonucleotides and other biologicalpolymers on surfaces. Such methods may be used to identify CRCA-1translation product ligands.

[0378] U.S. Pat. No. 5,324,483 issued to Cody et al. on Jun. 28, 1994,which is incorporated herein by reference, refers to apparatus formultiple simultaneous synthesis. The apparatus and method disclosedtherein may be used to produce multiple compounds which can be screenedto identify CRCA-1 translation product ligands.

[0379] U.S. Pat. No. 5,252,743 issued to Barrett et al. on Oct. 12,1993, which is incorporated herein by reference, refers to spatiallyaddressable immobilization of anti-ligands on surfaces. The methods andcompositions described therein may be used to identify CRCA-1translation product ligands.

[0380] U.S. Pat. No. 5,424,186 issued to Foder et al. on Jun. 13, 1995,which is incorporated herein by reference, refers to a very large scaleimmobilized polymer synthesis. The method of synthesizingoligonucleotides described therein may be used to identify CRCA-1translation product ligands.

[0381] U.S. Pat. No. 5,420,328 issued to Campbell on May 30, 1995, whichis incorporated herein by reference, refers to methods of synthesis ofphosphonate esters. The phosphonate esters so produced may be screenedto identify compounds which are CRCA-1 translation product ligands.

[0382] U.S. Pat. No. 5,288,514 issued to Ellman on Feb. 22, 1994, whichis incorporated herein by reference, refers to solid phase andcombinatorial synthesis of benzodiazepine compounds on a solid support.Such methods and compounds may be used to identify CRCA-1 translationproduct ligands.

[0383] As noted above, CRCA-1 translation product ligands may also beantibodies and fragments thereof. Indeed, antibodies raised to uniquedeterminants of these receptors will recognize that protein, and onlythat protein and, consequently, can serve as a specific targetingmolecule which can be used to direct novel diagnostics and therapeuticsto this unique marker. In addition, these antibodies can be used toidentify the presence of CRCA-1 translation product or fragments thereof in biological samples, to diagnose the presence of colorectal cancercells in vitro.

1. An in vitro method of determining whether or not an individual hasmetastasized colorectal cancer cells comprising the steps of examining asample of extraintestinal tissue and/or body fluids from an individualto determine whether CRCA-1 transcript is being expressed by cells insaid sample wherein expression of said CRCA-1 transcript is indicativeof the presence of metastasized colorectal cancer cells in said sample.2. The method of claim 1 wherein expression of said CRCA-1 transcript bysaid cells is determined by immunoassay wherein said sample is contactedwith detectable antibodies that specifically bind to a CRCA-1translation product.
 3. The method of claim 1 wherein expression of saidCRCA-1 transcript by said cells is determined by polymerase chainreaction wherein said sample is contacted with primers that selectivelyamplify said CRCA-1 transcript or cDNA generated therefrom.
 4. An invitro method of determining whether or not a tumor cell is a colorectaltumor cell comprising the steps of determining whether said tumor cellexpresses CRCA-1 transcript wherein expression of CRCA-1 transcriptindicates that the tumor cell is a colorectal tumor cell.
 5. The methodof claim 4 wherein expression of said CRCA-1 transcript by said tumorcells is determined by immunoassay wherein said cell is contacted withdetectable antibodies that specifically bind to a CRCA-1 translationproduct.
 6. The, method of claim 4 wherein expression of said CRCA-1transcript by said tumor cells is determined by polymerase chainreaction wherein said cell is contacted with primers that selectivelyamplify said CRCA-1 transcript or cDNA generated therefrom.
 7. An invitro method of determining whether or not an individual hasmetastasized colorectal cancer comprising the steps of examining asample of extraintestinal tissue and/or body fluids from an individualto determine whether a CRCA-1 translation product is present in saidsample, wherein the presence of a CRCA-1 translation product in saidsample indicates that said individual has metastasized colorectalcancer.
 8. The method of claim 7 wherein said CRCA-1 translation productis detected by immunoassay wherein said sample is contacted withdetectable antibodies that specifically bind to a CRCA-1 tranlationproduct.
 9. The method of claim 8 wherein said sample is body fluid. 10.The method of claim 9 wherein said sample is blood.
 11. An in vitromethod of determining whether or not an individual has metastasizedcolorectal cancer comprising the steps of examining a sample ofextraintestinal tissue and/or body fluids from an individual todetermine whether a CRCA-1 transcript is present in said sample, whereinthe presence of a CRCA-1 translation product in said sample indicatesthat said individual has metastasized colorectal cancer.
 12. The methodof claim 11 wherein said CRCA-1 transcript is detected by polymerasaechain reaction assay using primers which specifically amplify CRCA0-1transcript sequences.
 13. An in vitro assay kit for determining whetheror not an individual has metastasized colorectal cancer by detecting thepresence of a CRCA-1 translation product in a sample of extraintestinaltissue and/or body fluids from an individual, the presence of CRCA-1translation product is present in said sample, wherein the presence ofCRCA-1 translation product in said sample indicates that individual hasmetastasized colorectal cancer, said kit comprising: a containercomprising antibodies specific for a CRCA-1 translation product;instruction for using said kit.
 14. The kit of claim 13 furthercomprising a container that comprises a positive control; wherein saidpositive control is a sample containing a CRCA-1 translation productthat binds to said antibodies.
 15. The kit of claim 13 furthercomprising a container that comprises negative control; wherein saidnegative control is a sample free of said CRCA-1 translation product.16. The kit of claim 13 further comprising a container that comprises adetectable antibody that binds to said antibodies specific for saidCRCA-1 translation product.
 17. An in vitro PCR assay kit fordetermining whether or not an individual has colorectal cancer bydetecting the presence of CRCA-1 transcript in a sample ofextraintestinal tissue and/or body fluids from an individual, whereinthe presence of said CRCA-1 transcript in said sample indicates thatindividual has colorectal cancer, said kit comprising: a first containercomprising PCR primers that specifically amplify said CRCA-1 transcriptor cDNA generated therefrom; a second container comprising a sizemarker, said size marker being the expected size of amplified DNA ifsaid CRCA-1 transcript is present in said sample; and instructions forusing said kit.
 18. A substantially pure protein having an amino acidsequence selected from the group consisting of SEQ ID NO:2-81 andfunctional fragments thereof.
 19. The protein of claim 18 wherein saidprotein has an amino acid sequence selected from the group consisting ofSEQ ID NO:2-81.
 20. An isolated antibody which binds to an epitope on aprotein of claim
 19. 21. The antibody of claim 20 wherein said antibodyis a monoclonal antibody.
 22. An isolated nucleic acid molecule thatcomprises a nucleic acid sequence that encodes a protein of claim 18.23. The nucleic acid molecule of claim 22 wherein said protein encodedby said nucleic acid sequence has an amino acid sequence selected fromthe group consisting of SEQ ID NO:2-81.
 24. A recombinant expressionvector comprising the nucleic acid molecule of claim
 23. 25. A host cellcomprising the recombinant expression vector of claim
 24. 26. Anisolated nucleic acid molecule having a nucleic acid sequence of SEQ IDNO:1 or a functional fragment thereof.
 27. The nucleic acid molecule ofclaim 26 having a nucleic acid sequence of SEQ ID NO:1.
 28. Arecombinant expression vector comprising the nucleic acid molecule ofclaim
 27. 29. A host cell comprising the recombinant expression vectorof claim
 28. 30. The nucleic acid molecule of claim 26 consisting of afunctional fragment of SEQ ID NO:1 having at least 10 nucleotides. 31.The nucleic acid molecule of claim 26 consisting of a functionalfragment of SEQ ID NO:1 having 12-150 nucleotides.
 32. The nucleic acidmolecule of claim 26 consisting of a functional fragment of SEQ ID NO:1having 15-50 nucleotides.
 33. An oligonucleotide molecule comprising anucleotide sequence complimentary to a functional fragment of SEQ IDNO:1 having nucleotide sequence of at least 5 nucleotides.
 34. Theoligonucleotide molecule of claim 33 comprising a nucleotide sequencecomplimentary to a functional fragment of SEQ ID NO:1 having nucleotidesequence of 5-50 nucleotides.
 35. The oligonucleotide molecule of claim33 comprising a nucleotide sequence complimentary to a functionalfragment of SEQ ID NO:1 having nucleotide sequence of 10-40 nucleotides.36. The oligonucleotide molecule of claim 33 comprising a nucleotidesequence complimentary to a functional fragment of SEQ ID NO:1 havingnucleotide sequence of 15-25 nucleotides.
 37. The oligonucleotidemolecule of claim 33 comprising a nucleotide sequence complimentary to afunctional fragment of SEQ ID NO:1 having nucleotide sequence of 10-150nucleotides.
 38. The oligonucleotide molecule of claim 33 comprising anucleotide sequence complimentary to a functional fragment of SEQ IDNO:1 having nucleotide sequence of 18-28 nucleotides.
 39. A conjugatedcompound comprising: a) a CRCA-1 translation product binding moiety;and, b) an active moiety.
 40. The compound of claim 39 wherein saidactive moiety is a radiostable active agent.
 41. The compound of claim39 wherein said active moiety is radioactive.
 42. The compound of claim39 wherein said an active moiety is a therapeutic agent.
 43. Thecompound of claim 39 wherein said an active moiety is an imaging agent.44. The compound of claim 39 wherein said an active moiety is anantisense compound.
 45. The compound of claim 39 wherein said an activemoiety is selected from the group consisting of: methotrexate,doxorubicin, daunorubicin, cytosinarabinoside, etoposide, 5-4,fluorouracil, melphalan, chlorambucil, cis-platinum, vindesine,mitomycin, bleomycin, purothionin, macromomycin, 1,4-benzoquinonederivatives, trenimon, ricin, ricin A chain, Pseudomonas exotoxin,diphtheria toxin, Clostridium perfringens phospholipase C, bovinepancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin Achain, cobra venom factor, gelonin, saporin, modeccin, viscumin,volkensin, alkaline phosphatase, nitroimidazole, metronidazole,misonidazole, ⁴⁷Sc, ⁶⁷CU, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re,¹⁹⁹Au, ²¹¹At, ²¹²Pb, ²¹²B, ³²P and ³³P, ⁷¹Ge, ⁷⁷As ¹⁰³ Pb, ¹⁰⁵Rh, ¹¹¹Ag,¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os ^(193M)Pt, ¹⁹⁷Hg, ⁴³K,⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁸¹Rb/^(81M)Kr, ^(87M)Sr, ^(99M)Tc,¹¹¹In, ^(113M)In, ¹²³I, ¹²⁵I, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb and²⁰⁶Bi.
 46. The compound of claim 39 wherein said an active moiety is anantisense molecule that hybridizes to nucleotide sequences of DNA or RNAthat encode a gene selected from the group consisting of: hereditarynonpolyposis coli (HNPCC) genes such as hMSH2, hMLH1, hPMS1, and hPMS2,Ras, adenomatous polyposis coli (APC), ERBB-1/HER-1, ERBB-2/HER-2, p53Tumor Suppressor, MYB, FOS, ABL, MYC, Protein Tyrosine Phosphatase G1,Cyclic AMP-Dependent Protein Kinase (PKA), CRIPTO, Transforming GrowthFactor Alpha and 1p.
 47. A pharmaceutical composition comprising: a) apharmaceutically acceptable carrier or diluent, and, b) a compoundaccording to claim
 39. 48. A method of treating an individual suspectedof suffering from metastasized colorectal cancer comprising the steps ofadministering to said individual a pharmaceutical composition accordingto claim 47 wherein said active moiety is a therapeutic agent, saidcomposition is administered in an amount effective for therapeutic usein a humans suffering from colorectal cancer.
 49. A method ofradioimaging metastasized colorectal cancer cells comprising the stepsof administering to an individual a pharmaceutical composition accordingto claim 47 wherein said active moiety is an imaging agent, saidcomposition is administered in an amount effective for diagnostic use ina humans suffering from colorectal cancer.
 50. A method of treating anindividual suspected of suffering from colorectal cancer or ofpreventing colorectal cancer in an individual suspected of beingsusceptible to colorectal cancer comprising the steps of administeringto said individual a therapeutically or prophylactically effectiveamount of a pharmaceutical composition according to claim 47, whereinsaid active moiety is an antisense molecule that hybridizes tonucleotide sequences of DNA or RNA that encode a gene selected from thegroup consisting of: hereditary nonpolyposis coli (HNPCC) genes such ashMSH2, hMLH1, hPMS1, and hPMS2, Ras, adenomatous polyposis coli (APC),ERBB-1/HER-1, ERBB-2/HER-2, p53 Tumor Suppressor, MYB, FOS, ABL, MYC,Protein Tyrosine Phosphatase G1, Cyclic AMP-Dependent Protein Kinase(PKA), CRIPTO, Transforming Growth Factor. Alpha and 1p.
 51. The methodof claim 50 wherein said pharmaceutical composition is administeredorally.
 52. A method of delivery a nucleic acid molecule to intestinaltract cells of an individual comprising the steps of administering tosaid individual a pharmaceutical composition comprising: a) apharmaceutically acceptable carrier or diluent, and, b) a compositioncomprising: i) a CRCA-1 translation product ligand; and, ii) a nucleicacid molecule.
 53. A vaccine composition comprising: a) a proteincomprising at least one epitope of a CRCA-1 translation product or anucleic acid molecule that encodes said CRCA-1 translation product; andb) a pharmaceutically acceptable carrier or diluent.
 54. A method oftreating an individual who has metastasized colorectal cancer comprisingthe step of administering to such an individual a therapeuticallyeffective amount of a vaccine composition of claim
 53. 55. A method oftreating an individual who has been identified as being susceptible tometastasized colorectal cancer comprising the step of administering tosuch an individual a prophylactically effective amount of a vaccinecomposition of claim 53.